Variable region sequences of il-31 monoclonal antibodies and methods of use

ABSTRACT

Novel compositions derived from antigen-binding sites of immunoglobulins having affinity for IL-31 are provided. The compositions exhibit immunological binding properties of antibody molecules capable of binding specifically to a human IL-31. CDR regions derived from same or different immunoglobulin moieties are provided. Also provided are single chain polypeptides wherein V H  and V L  domains are attached. The sFv molecules can include ancillary polypeptide moieties which can be bioactive, or which provide a site of attachment for other useful moieties. The compositions are useful in specific binding assays, affinity purification schemes, drug or toxin targeting, imaging, and genetic or immunological therapeutics for inflammatory diseases. The invention thus provides novel polypeptides, the DNAs encoding those polypeptides, expression cassettes comprising those DNAs, and methods of inducing the production of the polypeptides. The invention further provides the amino acid sequences of the variable regions of the monoclonal antibodies and use of these monoclonal antibody or antibody fragment in conjunction with an human IgG4 Fc molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/895,008, filed May 15, 2013, which is a continuation of U.S.patent application Ser. No. 12/439,079, filed Feb. 26, 2009, now U.S.Pat. No. 8,466,262, which is the National Stage filed under 35 U.S.C.§371 of PCT Application No. PCT/US07/77555, filed Sep. 4, 2007, whichclaims benefit of U.S. Patent Application Ser. No. 60/890,792, filedFeb. 20, 2007, and U.S. Patent Application Ser. No. 60/824,403, filedSep. 1, 2006, all of which are herein incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

The skin plays an important role in the immune system and consists oflayers. The epidermis is a surface layer. Underneath the epidermis isthe dermis, a layer of connective tissue. Underneath the dermis, is thehypodermis, a layer of large amounts of adipose tissue. Circulating Tlymphocytes migrate to the skin under normal and inflammatoryconditions. The cutaneous lymphocyte antigen (CLA) is considered ahoming receptor for T cells with tropism for the skin. Santamaria-Babi,L., Eur. J. Dermatol. 14:13-18, 2004.

Several diseases of the skin are known to express high levels of CLA+ Tcells, including atopic dermatitis, contact dermatitis, drug-inducedallergic reactions, skin-tropic viruses and viral associated pruritis,vitiligo, cutaneous T cell lymphoma, alopecia aerata, acne rosacea, acnevulgaris, prurigo nodularis, and bullous pemphigoid. There is a need totreat such skin T cell mediated diseases.

The demonstrated in vivo activities of the cytokine family illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. IL-31, a newly identifiedcytokine. IL-31, when over-expressed in mice, results in dermatitis-likesymptoms. Both skin-homing T cells and epidermal keratinocytes have beenimplicated in the pathology of skin diseases in humans. The presentinvention addresses these needs by providing antagonists topro-inflammatory cytokine IL-31. Such antagonists of the presentinvention, which may block, inhibit, reduce, antagonize or neutralizethe activity of IL-31, include soluble IL-31RA receptors andneutralizing anti-IL-31 antibodies. The invention further provides usestherefore in inflammatory disease, as well as related compositions andmethods.

Monoclonal antibody technology has provided a vast array of therapeuticsas well as diagnostics for use in identifying and treating disease. Manyclinical applications have been focused on murine antihuman monoclonalantibodies, which are raised in mouse cells but which specifically binda human antigen. In addition, chimeric antibodies composed of human andnon-human amino acid sequences are being developed. Particularly, hybridantibody molecules having variable regions derived from, for example, amurine immunoglobulin fused to constant regions derived from a humanimmunoglobulin have been described. See e.g., U.S. Pat. No. 4,816,567;Winter et al. (1991) Nature 349:293-299; and Lobuglio et al. (1989)Proc. Nat. Acad. Sci. USA 86:4220-4224. Further, since constant regionsare not required for antigen recognition or binding, antibody fragmentssuch as F(ab), F(ab′)₂ and Fv which do not comprise the Fc portion havebeen indicated as useful candidates for clinical therapy.

A number of recombinant or biosynthetic molecules comprising rodentantigen-binding sites have been described. Particularly, moleculeshaving rodent antigen-binding sites built directly onto human antibodiesby grafting only the rodent binding site, rather than the entirevariable domain, into human immunoglobulin heavy and light chain domainshave been described. See, e.g., Riechmann et al. (1988) Nature332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536. Moleculeshaving an antigen-binding site wherein at least one of thecomplementarity determining regions (CDRS) of the variable domain isderived from a murine monoclonal antibody and the remainingimmunoglobulin-derived parts of the molecule are derived from humanimmunoglobulin have been described in U.K Patent Publication No. GB2,276,169, published Sep. 21, 1994. A number of single chainantigen-binding site polypeptides and single chain Fv (sFv) moleculeshave also been described. See, e.g., U.S. Pat. Nos. 5,132,405 and5,091,513 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

The effector function(s) of the Fc domain of an antibody includephagocytosis, release of inflammatory mediators, regulation of antibodyproduction, and most importantly antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Thedegree with which any of these effector functions are induced depends onthe interaction of the Fc domain with the relevant protein mediators,the Fcγ receptors and C1q, and differs depending on the IgG subclassconstant regions (Fc) and their interaction with these proteins.

The Fc domain of IgG1 interacts with FcγRI, FcγRIIa, and FcγRIII onimmune system effector cells. The precise role of the different Fcγreceptors remains to be elucidated but FcγRIIIA is thought to be themost important ADCC mediating receptor expressed primarily on NK cellsbut also monocytes and macrophages. IgG1 also binds C1q and can triggerCDC which is mediated principally by NK cells expressing FcγRIII. TheIgG4 Fc domain has largely reduced binding affinity to the different Fcγreceptors and C1q, corresponding to reduced ADCC and CDC. While IgG1shows generally high activity towards both ADCC and CDC, IgG4 isregarded as having low to no ADCC or CDC activity.

The binding affinity of the effector negative IgG4 mAb is greatlyreduced if not abolished when compared to other IgG isotype mAbs.However, the ability to interact with the Brambell receptor (FcRn) isretained in IgG4 thus affecting the pharmacokinetics of the IgG4 isotypemAb through an increased half-life.

Thus, there is a need for molecules which provide the amino acidsequences of anti-human IL-31 in conjunction with a human IgG4 Fcmolecule for treating IL-31 mediated inflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of the amino acid sequences of the variabledomains from the light chains and heavy chains of clones 292.12.3.1,292.84.1.6, 292.63.5.3, and 294.144.3.5.

FIG. 2 is an alignment of the amino acid sequences of the variabledomains from the light chains and heavy chains of clones 292.12.3.1 and292.84.1.6.

FIGS. 3 and 4 are an alignment of the amino acid sequences of the lightand heavy variable regions of mouse anti-human IL-31 monoclonalantibodies.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′)₂, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), whichare incorporated herein by reference). (See, generally, Hood et al.,“Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,Nature, 323, 15-16 (1986), which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994); see also International Patent Application PublicationNo. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, thedisclosures of which are incorporated by reference for any purpose. Inspecific embodiments, single-chain antibodies can also be bi-specificand/or humanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the determination of theamino acid sequences of monoclonal antibodies that were described inco-owned U.S. patent application Ser. No. 11/430,066, filed May 8, 2006,which was published on Dec. 7, 2006 as U.S. Patent Publication No.2006-0275296, incorporated herein by reference. Hybridomas expressingthe neutralizing monoclonal antibodies to human IL-31 described abovewere deposited with the American Type Tissue Culture Collection (ATCC;10801 University Blvd, Manassas Va. 20110-2209) patent depository asoriginal deposits under the Budapest Treaty and were given the followingATCC Accession No.s: clone 292.12.3.1 (ATCC Patent Deposit DesignationPTA-6815, deposited Jun. 29, 2005); clone 292.72.3.1 (ATCC PatentDeposit Designation PTA-6816, deposited Jun. 29, 2005); clone 292.63.5.3(ATCC Patent Deposit Designation PTA-6829, deposited Jul. 6, 2005);clone 292.118.6.4 (ATCC Patent Deposit Designation PTA-6830, depositedJul. 6, 2005); clone 294.163.2.1 (ATCC Patent Deposit DesignationPTA-6831, deposited Jul. 6, 2005); clone 292.84.1.6 (ATCC Patent DepositDesignation PTA-6871, deposited Jul. 19, 2005); clone 294.35.2.6.3 (ATCCPatent Deposit Designation PTA-6872, deposited Jul. 19, 2005); clone294.154.5.6 ATCC Patent Deposit Designation PTA-6875, deposited Jul. 19,2005); and clone 294.144.3.5 (ATCC Patent Deposit Designation PTA-6873,deposited Jul. 19, 2005).

The present invention provides the amino acid sequences of the light andheavy chain variable regions of the monoclonal antibodies produced bythese hybridomas to be used to generate antibodies and antibodyfragments, which bind to the IL-31 ligand and can be used in conjunctionwith an human IgG4 Fc molecule, for example by expression as a fusionprotein, to antagonize IL-31 thereby inhibiting, blocking, reducing, orneutralizing inflammation in general, and the symptoms of dermatitis andpruritic diseases. Such antibodies can comprise antibodies or antibodyfragments, comprising or consisting of a light chain variable region anda heavy chain variable region, and can be chimeric, humanized, orantibody fragments that neutralize, inhibit, reduce, prevent or minimizethe effects of IL-31 on its receptor. Clinical outcomes of the antibodyor antibody fragments can be a reduction in inflammatory diseases, suchas dermatitis and pruritic diseases as further described herein. In anembodiment, the dermatitis is atopic dermatitis. In another embodimentthe dermatitis is prurigo nodularis. In another embodiment, thedermatitis is eczema.

IL-31 is a recently discovered T cell cytokine which, whenover-expressed in mice, results in dermatitis-like symptoms. See also,Dillon, et al., Nature Immunol 5:752-760, 2004. Both skin-homing T cellsand epidermal keratinocytes have been implicated in the pathology ofskin diseases in humans. IL-31 mRNA and protein expression is restrictedto the skin-homing CLA+ T cell population in both atopic dermatitis (AD)patients and normal individuals, while analysis of the receptor forIL-31, IL-31RA, by immunohistochemistry (IHC) suggests slightly higherlevels of IL-31RA expression on skin keratinocytes in skin biopsies fromacute and chronic AD sufferers compared to normal individuals.

IL-31 is the HUGO name for a cytokine that has been previously describedas Zcyto17rlig in a published U.S. patent application (See publicationnumber 20030224487, Sprecher, Cindy et al., 2003, incorporated herein byreference). See also, Dillon, et al., Nature Immunol, supra. Theheterodimeric receptor for IL-31 was also described in 20030224487 aszcytor17 (HUGO name, IL-31RA) which forms a heterodimer with OncostatinMreceptor beta (OSMRbeta). IL-31 was isolated from a cDNA librarygenerated from activated human peripheral blood cells (hPBCs), whichwere selected for CD3. CD3 is a cell surface marker unique to cells oflymphoid origin, particularly T cells. The polypeptide sequence forhuman IL-31 is shown in SEQ ID NO:2. The polypeptide sequence for murineIL-31 is shown in SEQ ID NO:4. As used herein the term, IL-31 meansIL-31 as used in U.S. patent publication number 20030224487, as shownabove. The secretory signal sequence of IL-31 is comprised of amino acidresidues 1 (Met) to 23 (Ala), and the mature polypeptide is comprised ofamino acid residues 24 (Ser) to 164 (Thr) (as shown in SEQ ID NO:2).Further N-terminal sequencing analysis of purified IL-31 from 293T cellsshowed an N-terminus at residue 27 (Leu) as shown in SEQ ID NO:2, withthe mature polypeptide comprised of amino acid residues 27 (Leu) to 164(Thr) (as shown in SEQ ID NO:2).

IL-31 is the HUGO name for a cytokine that has been previously describedas Zcyto17rlig in a published U.S. patent application (See publicationnumber 20030224487, Sprecher, Cindy et al., 2003, incorporated herein byreference). See also, Dillon, et al., Nature Immunol, supra. Theheterodimeric receptor for IL-31 was also described in 20030224487 aszcytor17 (HUGO name, IL-31RA) which forms a heterodimer with OncostatinMreceptor beta (OSMRbeta). IL-31 was isolated from a cDNA librarygenerated from activated human peripheral blood cells (hPBCs), whichwere selected for CD3. CD3 is a cell surface marker unique to cells oflymphoid origin, particularly T cells. The polynucleotide andpolypeptide sequences for human IL-31 are shown in SEQ ID NOs: 1 and 2,respectively. The polynucleotide and polypeptide sequences for murineIL-31 are shown in SEQ ID NOs: 3 and 4, respectively. As used herein theterm, IL-31 means IL-31 as used in U.S. patent publication number20030224487, as shown above. The secretory signal sequence of IL-31 iscomprised of amino acid residues 1 (Met) to 23 (Ala), and the maturepolypeptide is comprised of amino acid residues 24 (Ser) to 164 (Thr)(as shown in SEQ ID NO:2). Further N-terminal sequencing analysis ofpurified IL-31 from 293T cells showed an N-terminus at residue 27 (Leu)as shown in SEQ ID NO:2, with the mature polypeptide comprised of aminoacid residues 27 (Leu) to 164 (Thr) (as shown in SEQ ID NO:2).

The polypeptide sequence for the IL-31RA (IL-31 receptor) is shown inSEQ ID NO:5, and the polypeptide sequence for OncostatinM receptor beta(OSMRbeta) is shown in SEQ ID NO:6.

The IL-31RA and OSMRbeta receptors belong to the Class I cytokinereceptor subfamily that includes, but is not limited to, the receptorsfor IL-2, IL-4, IL-7, Lif, IL-12, IL-15, EPO, TPO, GM-CSF and G-CSF (fora review see, Cosman, “The Hematopoietin Receptor Superfamily” inCytokine 5(2): 95-106, 1993). The IL-31RA subunit is fully described incommonly-owned PCT Patent Application No. US01/20484 (WIPO publicationNo. WO 02/00721). Analysis of the tissue distribution of the mRNA of theIL-31RA subunit revealed expression in activated CD4+ and CD8+ T-cellsubsets, CD14+ monocytes, and weaker expression in CD19+ B-cells.Moreover, the mRNA was present in both resting or activated monocyticcell lines THP-1 (ATCC No. TIB-202), U937 (ATCC No. CRL-1593.2) and HL60(ATCC No. CCL-240).

Inhibition, neutralization, or blocking signal transduction by themolecules comprising a light chain variable domain and or a heavy chainvariable domain, termed “IL-31 binding molecules” or “IL-31 antagonists”herein, can be measured by a number of assays known to one skilled inthe art. For example, assays measuring a reduction in proliferationinclude assays for reduction of a dye such as AlamarBlue™ (AccuMedInternational, Inc. Westlake, Ohio),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Mosman,J. Immunol Meth. 65: 55-63, 1983); 3,(4,5 dimethylthiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide; and cyanoditolyl-tetrazolium chloride (which are commerciallyavailable from Polysciences, Inc., Warrington, Pa.); mitogenesis assays,such as measurement of incorporation of ³H-thymidine; dye exclusionassays using, for example, naphthalene black or trypan blue; dye uptakeusing diacetyl fluorescein; and chromium release. See, in general,Freshney, Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,Wiley-Liss, 1994, which is incorporated herein by reference. In additionto the above, see published U.S. patent publication number 20030224487,(Sprecher, Cindy et al., 2003) for an example of of BaF3 cellsexpressing IL-31RA and full-length OSMRbeta.

Methods for preparing the polynucleotides encoding the antibodiesdescribed herin (including DNA and RNA) are well known in the art. TotalRNA can be prepared using guanidinium isothiocyanate extraction followedby isolation by centrifugation in a CsCl gradient (Chirgwin et al.,Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNAusing the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. In the alternative, genomic DNA can beisolated. Polynucleotides encoding IL-31 antibodies are then identifiedand isolated by, for example, hybridization or PCR.

The present invention also includes IL-31 binding molecules or IL-31antagonists that bind functional fragments of IL-31 polypeptides andnucleic acid molecules encoding such functional fragments. A“functional” IL-31 or fragment thereof as defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-IL-31 antibody or IL-31RA orantibody or IL-31RA/OSMRbeta heterodimers of these receptors (eithersoluble or immobilized). As previously described herein, IL-31 ischaracterized by a four-helical-bundle structure comprising helix A(amino acid residues 38-52), helix B (amino acid residues 83-98), helixC (amino acid residues 104-117) and helix D (amino acid residues137-152), as shown in SEQ ID NO:2. Thus, the present invention furtherprovides fusion proteins encompassing: (a) polypeptide moleculescomprising one or more of the helices described above; and (b)functional fragments comprising one or more of these helices. The otherpolypeptide portion of the fusion protein may be contributed by anotherfour-helical-bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF, orby a non-native and/or an unrelated secretory signal peptide thatfacilitates secretion of the fusion protein.

The present invention also provides IL-31 binding molecules or IL-31antagonists that bind to polypeptide fragments or peptides comprising anepitope-bearing portion of a IL-31 polypeptide described herein. Suchfragments or peptides may comprise an “immunogenic epitope,” which is apart of a protein that elicits an antibody response when the entireprotein is used as an immunogen Immunogenic epitope-bearing peptides canbe identified using standard methods (see, for example, Geysen et al.,Proc. Nat'l Acad. Sci. USA 81:3998 (1983)). The binding of theantibodies to these functional fragments results in inhibition,blocking, neutralization, and/or reduction in signal transduction ofIL-31 on its cognate receptor.

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodies(e.g., neutralizing antibodies) that bind with the polypeptidesdescribed herein. Hopp/Woods hydrophilicity profiles can be used todetermine regions that have the most antigenic potential (Hopp et al.,1981, ibid. and Hopp, 1986, ibid.). For example, in human IL-31,hydrophilic regions include amino acid residues 54-59 of SEQ ID NO:2,amino acid residues 129-134 of SEQ ID NO:2, amino acid residues 53-58 ofSEQ ID NO:2, amino acid residues 35-40 of SEQ ID NO:2, and amino acidresidues 33-38 of SEQ ID NO:2. For example, in mouse IL-31, hydrophilicregions include amino acid residues 34-39 of SEQ ID NO:4, amino acidresidues 46-51 of SEQ ID NO:4, amino acid residues 131-136 of SEQ IDNO:4, amino acid residues 158-163 of SEQ ID NO:4, and amino acidresidues 157-162 of SEQ ID NO:4.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fourteen amino acids,or about fourteen to about thirty amino acids of SEQ ID NO:2 or SEQ IDNO:4. Such epitope-bearing peptides and polypeptides can be produced byfragmenting a IL-31 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

The activity of the antibodies as described herein can be measured bytheir ability to inhibit, or reduce proliferation using a variety ofassays that measure proliferation of and/or binding to cells expressingthe IL-31RA receptor. Of particular interest are changes inIL-31-dependent cells. Suitable cell lines to be engineered to beIL-31-dependent include the IL-3-dependent BaF3 cell line (Palacios andSteinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell.Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790,1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growthfactor-dependent cell lines can be established according to publishedmethods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexteret al., in Baum et al. Eds., Experimental Hematology Today, 8th Ann.Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).

The activity of the anti-IL-31 antibodies described herein can bemeasured by a silicon-based biosensor microphysiometer which measuresthe extracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol Meth. 212:49-59, 1998;Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Antagonists are useful to inhibitexpansion, proliferation, activation, and/or differentiation of cellsinvolved in regulating hematopoiesis Inhibitors of IL-31 activity (IL-31antagonists) include anti-IL-31 antibodies and soluble IL-31 receptors,as well as other peptidic and non-peptidic agents (including ribozymes).

Inhibition the activity of IL-31 can be measured by a number of assays.In addition to those assays disclosed herein, samples can be tested forinhibition of IL-31 activity within a variety of assays designed tomeasure receptor binding, the stimulation/inhibition of IL-31-dependentcellular responses or proliferation of IL-31 RA receptor-expressingcells.

A IL-31-binding polypeptide, including IL-31 binding molecules or IL-31antagonists can also be used for purification of ligand. The polypeptideis immobilized on a solid support, such as beads of agarose,cross-linked agarose, glass, cellulosic resins, silica-based resins,polystyrene, cross-linked polyacrylamide, or like materials that arestable under the conditions of use. Methods for linking polypeptides tosolid supports are known in the art, and include amine chemistry,cyanogen bromide activation, N-hydroxysuccinimide activation, epoxideactivation, sulfhydryl activation, and hydrazide activation. Theresulting medium will generally be configured in the form of a column,and fluids containing ligand are passed through the column one or moretimes to allow ligand to bind to the receptor polypeptide. The ligand isthen eluted using changes in salt concentration, chaotropic agents(guanidine HCl), or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol Methods 145:229-40, 1991and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A receptor,antibody, member or fragment is covalently attached, using amine orsulfhydryl chemistry, to dextran fibers that are attached to gold filmwithin the flow cell. A test sample is passed through the cell. If aligand, epitope, or opposite member of the complement/anti-complementpair is present in the sample, it will bind to the immobilized receptor,antibody or member, respectively, causing a change in the refractiveindex of the medium, which is detected as a change in surface plasmonresonance of the gold film. This system allows the determination of on-and off-rates, from which binding affinity can be calculated, andassessment of stoichiometry of binding. Alternatively, ligand/receptorbinding can be analyzed using SELDI™ technology (Ciphergen, Inc., PaloAlto, Calif.).

IL-31 binding molecules or IL-31 antagonists can be used to block thebiological action of pro-inflammatory IL-31 and are useful asanti-inflammatory therapeutics in a variety of diseases as describedherein. One of skill in the art would recognize that antigenic,epitope-bearing polypeptides contain a sequence of at least 6,preferably at least 9, and more preferably at least 15 to about 30contiguous amino acid residues of a IL-31 polypeptide (e.g., SEQ IDNO:2). Polypeptides comprising a larger portion of a IL-31 polypeptide,i.e., from 30 to 100 residues up to the entire length of the amino acidsequence are included. Antigens or immunogenic epitopes can also includeattached tags, adjuvants, vehicles and carriers, as described herein.Suitable antigens include the IL-31 polypeptide encoded by SEQ ID NO:2from amino acid number 24 to amino acid number 164, or a contiguous 9 to141 amino acid fragment thereof. Other suitable antigens include, thefull length and the mature IL-31, helices A-D, and individual ormultiple helices A, B, C, and D, of the IL-31 four-helical-bundlestructure, as described herein. Preferred peptides to use as antigensare hydrophilic peptides such as those predicted by one of skill in theart from a hydrophobicity plot, as described herein, for example, aminoacid residues 114-119, 101-105, 126-131, 113-118, and 158-162 of SEQ IDNO:2; and amino acid residues 34-39, 46-51, 131-136, 158-163 and 157-162of SEQ ID NO:4. Moreover, IL-31 antigenic epitopes as predicted by aJameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc.,Madison, Wis.) serve as preferred antigens, and are readily determinedby one of skill in the art.

IL-31 binding molecules or IL-31 antagonists are considered to bespecifically binding if: 1) they exhibit a threshold level of bindingactivity, and 2) they do not significantly cross-react with relatedpolypeptide molecules. A threshold level of binding is determined ifIL-31 binding molecules or IL-31 antagonists herein bind to a IL-31polypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-IL-31) polypeptide. Itis preferred that the antibodies exhibit a binding affinity (K_(a)) of10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater. The bindingaffinity of IL-31 binding molecules or IL-31 antagonists can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672,1949).

Whether IL-31 binding molecules or IL-31 antagonists do notsignificantly cross-react with related polypeptide molecules is shown,for example, by the IL-31 binding molecules or IL-31 antagonistsdetecting IL-31 polypeptide but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are those disclosed in the prior art, such asknown orthologs, and paralogs, and similar known members of a proteinfamily. Screening can also be done using non-human IL-31, and IL-31mutant polypeptides. Moreover, IL-31 binding molecules or IL-31antagonists can be “screened against” known related polypeptides, toisolate a population that specifically binds to the IL-31 polypeptides.For example, IL-31 binding molecules or IL-31 antagonists are adsorbedto related polypeptides adhered to insoluble matrix; IL-31 bindingmolecules or IL-31 antagonists specific to IL-31 will flow through thematrix under the proper buffer conditions. Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988; Current Protocols in Immunology, Cooligan, et al. (eds.), NationalInstitutes of Health, John Wiley and Sons, Inc., 1995.

Monoclonal antibodies purified from tissue culture media arecharacterized for their ability to block or reduce the receptor bindingactivity (“neutralization assay”) of purified recombinant huIL-31 onBaF3/MPL-IL-31 cells.

Binding affinity of the IL-31 binding molecules and IL-31 antagonist canbe determined Goat-anti-Rat IgG-Fc gamma specific Antibody (Jackson) isimmobilized onto a CM5 Biacore chip. The assay is optimized to bind eachmAb onto the anti-Rat capture surface and then a concentration series ofIL-31 is injected across the mAb to see association (Ka) anddissociation (Kd). After each run, the surface is regenerated back tothe anti-Rat Antibody with 2 injections of 20 mM HCl. Data is generatedfor each and evaluation software (BlAevaluation software version 3.2,Pharmacia BIAcore, Uppsala, Sweden) is used to assess the kinetics ofthe anti-IL-31 antibody binding to the IL-31 protein.

The polynucleotide and polypeptide sequence of the light chain and heavychain variable regions of clones numbers 292.12.3.1, 292.84.1.6,292.63.5.3, 294.144.3.5, 292.39.5.3, 292.51.5.2, 292.64.6.5.5,292.105.4.1, 292.109.4.4, 292.118.6.4, 292.72.3.1. were determined asshown in Example 1. The polypeptide sequence of the amino terminus ofthe light and heavy chain variable regions of clones numbers weredetermined as shown in Example 2.

IL-31 binding molecules or IL-31 antagonists in tissue culture media arecharacterized for their ability to block, inhibit, prevent, or reducereceptor binding when grown in the presence of the purified recombinantproteins human IL-31. For example, the IL-31 binding molecules or IL-31antagonists can be characterized in a number of ways including binning(i.e., determining if each antibody could inhibit the binding of anyother binding), relative affinity, and neutralization.

IL-31 binding molecules or IL-31 antagonists generated by the methodsdescribed herein can be tested for neutralization by a variety ofmethods. For example the luciferase assay as described in published U.S.patent application (See publication number 20030224487, Sprecher, Cindyet al., 2003) can be used. In addition neutralization can be tested bymeasuring a decrease in the production of pro-inflammatory chemokinessuch as TARC and MDC from keratinocyte cultures in the presence ofligand and the monoclonal antibody. Neutralization can also be measuredby the in vivo models described herein.

In one embodiment, the IL-31 binding molecules or IL-31 antagonists ofthe present invention are human antigen-binding antibody fragments ofthe present invention and include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VHdomain. Antigen-binding antibody fragments, including single-chainantibodies, may comprise the variable region(s) (i.e., SEQ ID NO: 1, 2,3, or 4) alone or in combination with the entirety or a portion of thefollowing: hinge region, C_(H1), C_(H2), and C_(H3) domains. Alsoincluded in the invention are antigen-binding fragments also comprisingany combination of variable region(s) with a hinge region, C_(H1),C_(H2), and C_(H3) domains.

In another embodiment, the IL-31 binding molecules or IL-31 antagonistsof the present invention may be monospecific, bispecific, trispecific orof greater multispecificity. Multispecific antibodies may be specificfor different epitopes of a polypeptide of the present invention or maybe specific for both a polypeptide of the present invention as well asfor a heterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., PCT publications WO 93/17715; WO 92/08802;WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol 147:60-69 (1991);U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol 148:1547-1553 (1992).

The present invention also includes genetically altered IL-31 bindingmolecules or IL-31 antagonists that are functionally equivalent to theabove-described IL-31 binding molecules or IL-31 antagonists. ModifiedIL-31 binding molecules or IL-31 antagonists providing improvedstability and/or therapeutic efficacy are preferred. Examples ofmodified antibodies include those with conservative substitutions ofamino acid residues, and one or more deletions or additions of aminoacids which do not significantly deleteriously alter the antigen bindingutility. Substitutions can range from changing or modifying one or moreamino acid residues to complete redesign of a region as long as thetherapeutic utility is maintained. IL-31 binding molecules or IL-31antagonists of the present invention can be can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).

The IL-31 binding molecules or IL-31 antagonists also include chimericantibodies or chimeric fragments, comprising a variable region asdescribed herein and a constant region derived from a human so that thechimeric antibody or chimeric fragment has a longer half-life, and isless immunogenic when administered to a human subject. The method ofmaking chimeric antibodies and chimeric fragments is known in the art.The variable regions of these IL-31 binding molecules or IL-31antagonists can be connected with a constant region of a human IgG toform the desired chimeric antibody. An IgG Fc molecule can be fused tothe IL-31 binding molecules. To avoid induction of effector function,the IgG Fc molecule can be from an IgG4 Fc molecule. Thus, the aminoacid sequences of the variable regions and variable regions as disclosedherein can be used to generate chimeric antibodies wherein the constantregion is a human IgG molecule and the light chain and heavy chainvariable regions can be selected from those of clones 292.12.3.1,292.84.1.6, 292.63.5.3, 294.144.3.5, 292.39.5.3, 292.51.5.2,292.64.6.5.5, 292.105.4.1, 292.109.4.4, 292.118.6.4, and 292.72.3.1.Such chimeric antibodies would have: a) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 8 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 9; b) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 10 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 11; c) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 12 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 13; d) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 15; e) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 16 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 17; f) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 18 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 19; g) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 20 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 21; h) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 22 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:23; i) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 24 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 25; or j) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 26 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 27 andused in conjunction with an human IgG4 Fc molecule.

Such variable regions can also be generated with or without the signalsequences. Thus, the amino acid sequences of the variable regions andvariable regions as disclosed herein can be used to generate chimericantibodies wherein the constant region is a human IgG molecule and thelight chain and heavy chain variable regions can have a signal sequenceand can be selected from those of clones 292.12.3.1, 292.84.1.6,292.63.5.3, 294.144.3.5, 292.39.5.3, 292.51.5.2, 292.64.6.5.5,292.105.4.1, 292.109.4.4, 292.118.6.4, and 292.72.3.1. Such chimericantibodies would have: a) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 31 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 32; b) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 2 and aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:4; c) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 35 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 36; d) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 37 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:38; e) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 39 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 40; f) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 41 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 42; g)a light chain variable region comprising the amino acid sequence of SEQID NO: 43 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 44; h) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 45 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 46; i) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:47 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 48; or j) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 49 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 50 and used inconjunction with an human IgG4 Fc molecule. Such variable regions can begenerated with or without the signal sequences.

The IL-31 binding molecules or IL-31 antagonists include humanizedversion of the IL-31 binding molecules or IL-31 antagonists describedherein. Humanized IL-31 binding molecules or IL-31 antagonists compriseCDRs of a mouse donor immunoglobulin and heavy chain and light chainframeworks of a human acceptor immunoglobulin. The method of makinghumanized antibody is disclosed in U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762; and 6,180,370 (each of which is incorporated by reference inits entirety). The CDRs of these antibodies can then be grafted to anyselected human frameworks, which are known in the art, to generate thedesired humanized antibody.

The invention also provides IL-31 binding molecules or IL-31 antagoniststhat competitively inhibit the binding of a monoclonal antibody to apolypeptide of the invention, preferably the polypeptide of SEQ ID NO:2or SEQ ID NO:4. Competitive inhibition can be determined by any methodknown in the art, for example, using the competitive binding assaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits the binding of a monoclonal antibody of the invention by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50% tothe polypeptide of SEQ ID NO:2 or SEQ ID NO:4.

The invention also provides IL-31 binding molecules or IL-31 antagoniststhat competitively inhibit binding of an antibody to an epitope of theinvention as determined by any method known in the art for determiningcompetitive binding, for example, the immunoassays described herein. Inpreferred embodiments, the antibody competitively inhibits binding tothe epitope by at least 90%, at least 80%, at least 70%, at least 60%,or at least 50%.

The IL-31 binding molecules or IL-31 antagonists of the presentinvention include derivatives that are modified, for example, but not byway of limitation, the derivatives include IL-31 binding molecules orIL-31 antagonists that have been modified, e.g., by glycosylation,acetylation, pegylation, phosphylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

The IL-31 binding molecules or IL-31 antagonists of the presentinvention also encompass IL-31 binding molecules or IL-31 antagoniststhat have half-lives (e.g., serum half-lives) in a mammal, preferably ahuman, of greater than 15 days, preferably greater than 20 days, greaterthan 25 days, greater than 30 days, greater than 35 days, greater than40 days, greater than 45 days, greater than 2 months, greater than 3months, greater than 4 months, or greater than 5 months. The increasedhalf-lives of the antibodies of the present invention or fragmentsthereof in a mammal, preferably a human, result in a higher serum titerof said antibodies or antibody fragments in the mammal, and thus, reducethe frequency of the administration of said antibodies or antibodyfragments and/or reduces the concentration of said antibodies orantibody fragments to be administered.

The in vivo half-lives of the IL-31 binding molecules or IL-31antagonists can be increased by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631 and WO 02/060919, which are incorporatedherein by reference in their entireties), or by attaching polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N- or C-terminus of saidantibodies or antibody fragments or via epsilon-amino groups present onlysine residues. Linear or branched polymer derivatization that resultsin minimal loss of biological activity will be used. The degree ofconjugation will be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates by, e.g.,size exclusion or ion-exchange chromatography.

It is understood that the humanized IL-31 binding molecules or IL-31antagonists designed by the present method may have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. By conservativesubstitutions is intended combinations such as gly, ala; val, ile, leu;asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Methods for humanizing non-human antibodies are well known in the art.Generally, humanized immunoglobulins, including humanized antibodies,have been constructed by means of genetic engineering. Most humanizedimmunoglobulins that have been previously described (Jones et al., op.cit.; Verhoeyen et al., op. cit.; Riechmann et al., op. cit.) havecomprised a framework that is identical to the framework of a particularhuman immunoglobulin chain, the acceptor, and three CDR's from anon-human donor immunoglobulin chain. Specifically, humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule.

The present invention includes criteria by which a limited number ofamino acids in the framework of a humanized immunoglobulin chain arechosen to be the same as the amino acids at those positions in the donorrather than in the acceptor, in order to increase the affinity of anantibody comprising the humanized immunoglobulin chain.

The IL-31 binding molecules or IL-31 antagonists of the presentinvention are generated based in part on the model that two contributingcauses of the loss of affinity in prior means of producing humanizedantibodies (using as examples mouse antibodies as the source of CDR's)are:

(1) When the mouse CDR's are combined with the human framework, theamino acids in the framework close to the CDR's become human instead ofmouse. Without intending to be bound by theory, these changed aminoacids may slightly distort the CDR's, because they create differentelectrostatic or hydrophobic forces than in the donor mouse antibody,and the distorted CDR's may not make as effective contacts with theantigen as the CDR's did in the donor antibody; and

(2) Amino acids in the original mouse antibody that are close to, butnot part of, the CDR's (i.e., still part of the framework), may makecontacts with the antigen that contribute to affinity. These amino acidsare lost when the antibody is humanized, because all framework aminoacids are made human.

To avoid these problems, and to produce humanized antibodies that have avery strong affinity for a desired antigen, the present invention usesone or more of the following principles for designing humanizedimmunoglobulins. Also, the criteria may be used singly, or whennecessary in combination, to achieve the desired affinity or othercharacteristics.

A principle is that as acceptor, a framework is used from a particularhuman immunoglobulin that is unusually homologous to the donorimmunoglobulin to be humanized, or use a consensus framework from manyhuman antibodies. For example, comparison of the sequence of a mouseheavy (or light) chain variable region against human heavy (or light)variable regions in a data bank (for example, the National BiomedicalResearch Foundation Protein Identification Resource) shows that theextent of homology to different human regions varies greatly, typicallyfrom about 40% to about 60-70%. By choosing as the acceptorimmunoglobulin one of the human heavy (respectively light) chainvariable regions that is most homologous to the heavy (respectivelylight) chain variable region of the donor immunoglobulin, fewer aminoacids will be changed in going from the donor immunoglobulin to thehumanized immunoglobulin. Hence, and again without intending to be boundby theory, it is believed that there is a smaller chance of changing anamino acid near the CDR's that distorts their conformation. Moreover,the precise overall shape of a humanized antibody comprising thehumanized immunoglobulin chain may more closely resemble the shape ofthe donor antibody, also reducing the chance of distorting the CDR's.

Typically, one of the 3-5 most homologous heavy chain variable regionsequences in a representative collection of at least about 10 to 20distinct human heavy chains will be chosen as acceptor to provide theheavy chain framework, and similarly for the light chain. Preferably,one of the 1-3 most homologous variable regions will be used. Theselected acceptor immunoglobulin chain will most preferably have atleast about 65% homology in the framework region to the donorimmunoglobulin.

In many cases, it may be considered preferable to use light and heavychains from the same human antibody as acceptor sequences, to be surethe humanized light and heavy chains will make favorable contacts witheach other. In this case, the donor light and heavy chains will becompared only against chains from human antibodies whose completesequence is known, e.g., the Eu, Lay, Pom, Wol, Sie, Gal, Ou and WEAantibodies (Kabat et al., op. cit.; occasionally, the last few aminoacids of a human chain are not known and must be deduced by homology toother human antibodies). The human antibody will be chosen in which thelight and heavy chain variable regions sequences, taken together, areoverall most homologous to the donor light and heavy chain variableregion sequences. Sometimes greater weight will be given to the heavychain sequence. The chosen human antibody will then provide both lightand heavy chain acceptor sequences. In practice, it is often found thatthe human Eu antibody will serve this role.

According to the “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol, 151: 2296 (1993); Chothiaet al., J. Mol. Biol., 196: 901 (1987)). Another method uses aparticular framework derived from the consensus sequence of all humanantibodies of a particular subgroup of light or heavy chains. The sameframework may be used for several different humanized antibodies (Carteret al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J.Immnol, 151: 2623 (1993)).

Regardless of how the acceptor immunoglobulin is chosen, higher affinitymay be achieved by selecting a small number of amino acids in theframework of the humanized immunoglobulin chain to be the same as theamino acids at those positions in the donor rather than in the acceptor.Often, framework residues in the human framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species.

Humanized antibodies generally have at least three potential advantagesover mouse or in some cases chimeric antibodies for use in humantherapy:

(1) Because the effector portion is human, it may interact better withthe other parts of the human immune system (e.g., destroy the targetcells more efficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC)).

(2) The human immune system should not recognize the framework orconstant region of the humanized antibody as foreign, and therefore theantibody response against such an injected antibody should be less thanagainst a totally foreign mouse antibody or a partially foreign chimericantibody.

(3) Injected mouse antibodies have been reported to have a half-life inthe human circulation much shorter than the half-life of normalantibodies (D. Shaw et al., J. Immunol, 138, 4534-4538 (1987)). Injectedhumanized antibodies will presumably have a half-life more similar tonaturally occurring human antibodies, allowing smaller and less frequentdoses to be given.

In one aspect, the present invention is directed to designing humanizedantibodies that are produced by expressing recombinant DNA segmentsencoding the heavy and light chain CDR's from a donor immunoglobulincapable of binding to a desired antigen, such as IL-31. attached to DNAsegments encoding acceptor human framework regions.

The DNA segments will typically further include an expression controlDNA sequence operably linked to the humanized immunoglobulin codingsequences, including naturally-associated or heterologous promoterregions. The expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the humanized light chains, heavy chains,light/heavy chain dimers or intact antibodies, binding fragments orother immunoglobulin forms may follow (see, S. Beychok, Cells ofImmunoglobulin Synthesis, Academic Press, N.Y., (1979), which isincorporated herein by reference). The anti-IL_(—)31 molecules can bemade by expression in mammalian host cells, such as Chinese HamsterOvary cells, and HEK 293F cells, for example.

Human constant region DNA sequences can be isolated in accordance withwell known procedures from a variety of human cells, but preferablyimmortalized B-cells (see, Kabat op. cit. and WP87/02671). The CDR's forproducing the immunoglobulins of the present invention will be similarlyderived from monoclonal antibodies capable of binding to thepredetermined antigen, such as IL-31, and produced by well known methodsin any convenient mammalian source including, mice, rats, rabbits, orother vertebrates, capable of producing antibodies. Suitable sourcecells for the constant region and framework DNA sequences, and hostcells for immunoglobulin expression and secretion, can be obtained froma number of sources, such as the American Type Culture Collection(“Catalogue of Cell Lines and Hybridomas,” sixth edition (1988)Rockville, Md. U.S.A., which is incorporated herein by reference).

In addition to the humanized immunoglobulins specifically describedherein, other “substantially homologous” modified immunoglobulins to thenative sequences can be readily designed and manufactured utilizingvarious recombinant DNA techniques well known to those skilled in theart. A variety of different human framework regions may be used singlyor in combination as a basis for the humanized immunoglobulins of thepresent invention. In general, modifications of the genes may be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis (see, Gillman and Smith, Gene, 8, 81-97 (1979)and S. Roberts et al., Nature, 328, 731-734 (1987), both of which areincorporated herein by reference).

The humanized antibodies of the invention include fragments as well asintact antibodies. Typically, these fragments compete with the intactantibody from which they were derived for antigen binding. The fragmentstypically bind with an affinity of at least 10⁷ M.⁻¹, and more typically10⁸ or 10⁹ M.⁻¹ (i.e., within the same ranges as the intact antibody).Humanized antibody fragments include separate heavy chains, light chainsFab, Fab′ F(ab′)₂, and Fv. Fragments are produced by recombinant DNAtechniques, or by enzymic or chemical separation of intactimmunoglobulins.

For further details in humanizing antibodies, see European Patent Nos.EP 239,400, EP 592,106, and EP 519,596; International Publication Nos.WO 91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991,Molecular Immunology 28(4/5): 489 498; Studnicka et al., 1994, ProteinEngineering 7(6): 805 814; Roguska et al., 1994, PNAS 91: 969 973; Tanet al., 2002, J. Immunol 169: 1119 25; Caldas et al., 2000, Protein Eng.13: 353 60; Morea et al., 2000, Methods 20: 267 79; Baca et al., 1997,J. Biol. Chem. 272: 10678 84; Roguska et al., 1996, Protein Eng. 9: 895904; Couto et al., 1995, Cancer Res. 55 (23 Supp): 5973s 5977s; Couto etal., 1995, Cancer Res. 55: 1717 22; Sandhu, 1994, Gene 150: 409 10;Pedersen et al., 1994, J. Mol. Biol. 235: 959 73; Jones et al., 1986,Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329; andPresta, 1992, Curr. Op. Struct. Biol. 2: 593-596.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24: 107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′).sub.2 fragments (Carter etal., Bio/Technology 10: 163-167 (1992)). According to another approach,F(ab′).sub.2 fragments can be isolated directly from recombinant hostcell culture. Other techniques for the production of antibody fragmentswill be apparent to the skilled practitioner. Further, examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. Thus, the invention also pertains toimmunoconjugates comprising the antibody described herein conjugated toa cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) fused or conjugated to heterologouspolypeptide sequences (e.g., antibody domains other than the variableregions). For example, the polypeptides of the present invention may befused or conjugated to an antibody Fc region, or portion thereof. Forexample, polypeptides of the present invention (including fragments orvariants thereof), may be fused with the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (C_(H1),C_(H2), C_(H3), or any combination thereof and portions thereof,resulting in chimeric polypeptides. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, C_(H1) domain, C_(H2) domain, and C_(H3) domain or anycombination of whole domains or portions thereof. The polypeptides mayalso be fused or conjugated to the above antibody portions to formmultimers. For example, Fc portions fused to the polypeptides of thepresent invention can form dimers through disulfide bonding between theFc portions. Higher multimeric forms can be made by fusing thepolypeptides to portions of IgA and IgM. Methods for fusing orconjugating the polypeptides of the present invention to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al.,Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.Immunol 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties). By way of another non-limiting example, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused with albumin (including but not limited torecombinant human serum albumin or fragments or variants thereof (see,e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622,and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporatedby reference in their entirety)). Polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) may be fusedto either the N- or C-terminal end of the heterologous protein (e.g.,immunoglobulin Fc polypeptide or human serum albumin polypeptide).Polynucleotides encoding fusion proteins of the invention are alsoencompassed by the invention.

As discussed above, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (See, e.g., EP 394,827;Traunecker et al., Nature 331:84-86 (1988)). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0. Such techniques alsoinclude, but are not limited to, the use of bifunctional conjugatingagents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239;5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604;5,274,119; 4,994,560; and 5,808,003; the contents of each of which arehereby incorporated by reference in its entirety).

Moreover, the polypeptides of the invention (e.g., antibodies orfragments thereof) can be fused to marker sequences, such as a peptideto facilitates their purification. In a further embodiment, nucleicacids encoding the polypeptides of the invention (including, but notlimited to nucleic acids encoding immunogenic and/or antigenic epitopes)can also be recombined with a gene of interest as an epitope tag (e.g.,the hemagglutinin tag (“HA”) or flag tag) to aid in detection andpurification of the expressed polypeptide. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses IL-31 binding molecules orIL-31 antagonists conjugated to a diagnostic or therapeutic agent. TheIL-31 binding molecules or IL-31 antagonists can be used diagnosticallyto, for example, monitor the development or progression of a tumor aspart of a clinical testing procedure to, e.g., determine the efficacy ofa given treatment, diagnosis, detection, and/or prevention regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 formetal ions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, IL-31 binding molecules or IL-31 antagonists may be conjugatedto a therapeutic moiety such as a cytotoxin, e.g., a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g., angiostatin or endostatin; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophasecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other growth factors.

IL-31 binding molecules or IL-31 antagonists may also be attached tosolid supports, which are particularly useful for immunoassays orpurification of the target antigen. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol Rev. 62:119-58 (1982).

Alternatively, IL-31 binding molecules or IL-31 antagonists can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980, which is incorporatedherein by reference in its entirety.

An IL-31 binding molecules or IL-31 antagonists, with or without atherapeutic moiety conjugated to it, administered alone or incombination with cytotoxic factor(s) and/or cytokine(s) can be used as atherapeutic.

A variety of assays known to those skilled in the art can be utilized todetect binding of IL-31 binding molecules or IL-31 antagonists to IL-31proteins or polypeptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant IL-31 protein or polypeptide.

IL-31 binding molecules or IL-31 antagonists to IL-31 may be used fortagging cells that express IL-31; for isolating IL-31 by affinitypurification; for diagnostic assays for determining circulating levelsof IL-31 polypeptides; for detecting or quantitating soluble IL-31 as amarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies; and as neutralizing antibodies or asantagonists to block IL-31 activity in vitro and in vivo. Suitabledirect tags or labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent markers, chemiluminescent markers,magnetic particles and the like; indirect tags or labels may feature useof biotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to IL-31 or fragments thereof may be used in vitroto detect denatured IL-31 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

The choice of the Fc domain may have implications on (i) the antibody'seffector functions (FcγR binding (ADCC)/C1q binding (CDC)) andpotentially associated side-effects, (ii) its pharmacokinetic properties(FcRn binding) including half-life, and (iii) possibly on immune complexclearance. The effector function(s) of the Fc domain includephagocytosis, release of inflammatory mediators, regulation of antibodyproduction, and most importantly antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Thedegree with which any of these effector functions are induced depends onthe interaction of the Fc domain with the relevant protein mediators,the Fcγ receptors and C1q, and differs depending on the IgG subclassconstant regions (Fc) and their interaction with these proteins.

The Fc domain of IgG1 interacts with FcγRI, FcγRIIa, and FcγRIII onimmune system effector cells. The precise role of the different Fcγreceptors remains to be elucidated but FcγRIIIA is thought to be themost important ADCC mediating receptor expressed primarily on NK cellsbut also monocytes and macrophages. IgG1 also binds C1q and can triggerCDC which is mediated principally by NK cells expressing FcγRIII. SeeGessner, J. E., et al., The IgG Fc Receptor Family, Ann. Hematol. 1998June: 76(6): 231-248. The IgG4 Fc domain has largely reduced bindingaffinity to the different Fcγ receptors and C1q, corresponding toreduced ADCC and CDC. For example, Sharma et al. examined a directcomparison of an IgG1 mAb and its IgG4 derivative, each with identicalvariable regions that targeted CD4. Both mAbs decreased CD4 expressionat equal dose levels and had equivalent pharmacokinetic properties, butthe IgG1 form showed a more potent ADCC effect. See Sharma A., et al.,Comparative pharmacodynamics of keliximab and clenoliximab in transgenicmice bearing human CD4. J Pharmacol Exp Ther. 2000 April;293(1):33-41.

The binding affinity of the effector negative IgG4 mAb is greatlyreduced if not abolished when compared to other IgG isotype mAbs.However, the ability to interact with the Brambell receptor (FcRn) isretained in IgG4 thus affecting the pharmacokinetics of the IgG4 isotypemAb through an increased half-life. While IgG1 shows generally highactivity towards both ADCC and CDC, IgG4 is regarded as having low to noADCC or CDC activity.

Several examples of IgG4 isotype mAbs that are either on the market, inthe clinic or at various stages of research and development. Thesetherapeutic mAbs are developed for a variety of indications in oncology,autoimmunity & inflammation as well as in anti-viral therapy.

As reported by Aalberse et al. (See Aalberse R C, Schuurman, J. IgG4breaking the rules, Immunology 2002, 105: 9-19), human (IgG4) exists intwo molecular forms due to the heterogeneity of the inter-heavy chaindisulfide bridges in the hinge region in a portion of secreted humanIgG4. This heterogeneity is only revealed under denaturing, non-reducingconditions in which an HL “half antibody” is detected, a phenomenon notseen in other human IgG isotypes. Taylor report that artifactualphenomena during sample handling such as rapid disulfide scrambling andreoxidation upon exposure to reductant can contribute to the amount ofhalf-antibody present (Taylor F R, et al., Suppression of Sodium DodecylSulfate-Polyacrylamide Gel Electrophoresis Sample Preparation Artifactsfor Analysis of IgG4 Half-Antibody. Analytical Biochemistry 2006, 353:204-208). Under native conditions, noncovalent interactions ensure thatthe antibody is held together as the H2L2 tetramer. Though, IgG4 hasbeen reported to hetero-dimerize with other IgG4 molecules incirculation. Analysis of the hinge sequences that connect the F[ab] andFc portion of the antibody of human IgG heavy chains suggests that thepresence of serine at residue 241 might be the cause of thisheterogeneity: the IgG4 hinge region contains a Cys-Pro-Ser-Cys sequencerather than a Cys-Pro-Pro-Cys sequence as in IgG1. Changing the serineat 241 to proline (found at that position in IgG1 and IgG2) in amouse/human chimeric heavy chain leads to the production of ahomogeneous antibody and abolishes the heterogeneity. Further, thevariant IgG4 has significantly extended serum half-life and shows animproved tissue distribution compared to the original chimeric IgG4.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria, toxin,saporin, Pseudomonas exotoxin, ricin, abrin and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90(either directly attached to the polypeptide or antibody, or indirectlyattached through means of a chelating moiety, for instance).Polypeptides or antibodies may also be conjugated to cytotoxic drugs,such as adriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

IL-31 binding molecules or IL-31 antagonists can also act as IL-31“antagonists” to block IL-31 binding and signal transduction in vitroand in vivo. These IL-31 binding molecules or IL-31 antagonists would beuseful for inhibiting IL-31 activity or protein-binding.

Polypeptide-toxin fusion proteins or antibody-toxin fusion proteins canbe used for targeted cell or tissue inhibition or ablation (forinstance, to treat cancer cells or tissues). Alternatively, if thepolypeptide has multiple functional domains (i.e., an activation domainor a receptor binding domain, plus a targeting domain), a fusion proteinincluding only the targeting domain may be suitable for directing adetectable molecule, a cytotoxic molecule or a complementary molecule toa cell or tissue type of interest. In instances where the domain onlyfusion protein includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting carrier or vehicle for cell/tissue-specific delivery ofgeneric anti-complementary-detectable/cytotoxic molecule conjugates.

A further object of the present invention is an isolated nucleic acidmolecule encoding any of the antibodies here above or below described,or a complementary strand or degenerate sequence thereof. In thisregard, the term “nucleic acid molecule” encompasses all different typesof nucleic acids, including without limitation deoxyribonucleic acids(e.g., DNA, cDNA, gDNA, synthetic DNA, etc.), ribonucleic acids (e.g.,RNA, mRNA, etc.) and peptide nucleic acids (PNA). In a preferredembodiment, the nucleic acid molecule is a DNA molecule, such as adouble-stranded DNA molecule or a cDNA molecule. The term “isolated”means nucleic acid molecules that have been identified and separatedfrom at least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated nucleic acid molecules therefore are distinguished fromthe specific nucleic acid molecule as it exists in natural cells. Adegenerate sequence designates any nucleotide sequence encoding the sameamino acid sequence as a reference nucleotide sequence, but comprising adistinct nucleotide sequence as a result of the genetic code degeneracy.

A further object of this invention is a vector comprising DNA encodingany of the above or below described antibodies. The vector may be anycloning or expression vector, integrative or autonomously replicating,functional in any prokaryotic or eukaryotic cell. In particular, thevector may be a plasmid, cosmid, virus, phage, episome, artificialchromosome, and the like. The vector may comprise regulatory elements,such as a promoter, terminator, enhancer, selection marker, origin ofreplication, etc. Specific examples of such vectors include prokaryoticplasmids, such as pBR, pUC or pcDNA plasmids; viral vectors, includingretroviral, adenoviral or AAV vectors; bacteriophages; baculoviruses;BAC or YAC, etc., as will be discussed below. The appropriate nucleicacid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

A further aspect of the present invention is a recombinant host cell,wherein said cell comprises a nucleic acid molecule or a vector asdefined above. The host cell may be a prokaryotic or eukaryotic cell.Examples of prokaryotic cells include bacteria, such as E. coli.Examples of eucaryotic cells are yeast cells, plant cells, mammaliancells and insect cells including any primary cell culture or establishedcell line (e.g., 3T3, Véro, HEK293, TNS, etc.). Suitable host cells forthe expression of glycosylated proteins are derived from multicellularorganisms. Examples of invertebrate cells include insect cells such asDrosophila S2 and Spodoptera Sf9, as well as plant cells. Examples ofuseful mammalian host cell lines include Chinese hamster ovary (CHO) andCOS cells. More specific examples include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl, Acad. Sci. USA, 77:4216 (1980));mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980));human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). Particularlypreferred mammalian cells of the present invention are CHO cells.

As disclosed here above, the antibodies of the present invention may beproduced by any technique known per se in the art, such as byrecombinant technologies, chemical synthesis, cloning, ligations, orcombinations thereof. In a particular embodiment, the antibodies areproduced by recombinant technologies, e.g., by expression of acorresponding nucleic acid in a suitable host cell. Another object ofthis invention is therefore a method of producing an antibody of thepresent invention, the method comprising culturing a recombinant hostcell of the invention under conditions allowing expression of thenucleic acid molecule, and recovering the polypeptide produced. Thepolypeptide produced may be glycosylated or not, or may contain otherpost-translational modifications depending on the host cell type used.Many books and reviews provide teachings on how to clone and producerecombinant proteins using vectors and prokaryotic or eukaryotic hostcells, such as some titles in the series “A Practical Approach”published by Oxford University Press (“DNA Cloning 2: ExpressionSystems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996; “ProteinExpression”, 1999; “Protein Purification Techniques”, 2001).

The vectors to be used in the method of producing an antibody accordingto the present invention can be episomal or non-/homologouslyintegrating vectors, which can be introduced into the appropriate hostcells by any suitable means (transformation, transfection, conjugation,protoplast fusion, electroporation, calcium phosphate-precipitation,direct microinjection, etc.). Factors of importance in selecting aparticular plasmid, viral or retroviral vector include: the ease withwhich recipient cells that contain the vector may be recognized andselected from those recipient cells which do not contain the vector; thenumber of copies of the vector which are desired in a particular host;and whether it is desirable to be able to “shuttle” the vector betweenhost cells of different species. The vectors should allow the expressionof the polypeptide or fusion proteins of the invention in prokaryotic oreukaryotic host cells, under the control of appropriate transcriptionalinitiation/termination regulatory sequences, which are chosen to beconstitutively active or inducible in said cell. A cell linesubstantially enriched in such cells can be then isolated to provide astable cell line.

Host cells are transfected or transformed with expression or cloningvectors described herein for protein production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Preferred cells to be used in the present invention are eukaryotic hostcells, e.g. mammalian cells, such as human, monkey, mouse, and ChineseHamster Ovary (CHO) cells, because they provide post-translationalmodifications to protein molecules, including correct folding orglycosylation at correct sites. Examples of suitable mammalian hostcells include African green monkey kidney cells (Vero; ATCC CRL 1587),human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamsterkidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), caninekidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1;ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555,1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCCCCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformedmonkey kidney cells (COS-1; ATCC CRL 1650), Bowes melanoma and humanhepatocellular carcinoma (for example Hep G2), murine embryonic cells(NIH-3T3; ATCC CRL 1658) and a number of other cell lines. Alternativeeukaryotic host cells are yeast cells (e.g., Saccharomyces,Kluyveromyces, etc.) transformed with yeast expression vectors. Alsoyeast cells can carry out post-translational peptide modificationsincluding glycosylation. A number of recombinant DNA strategies existwhich utilize strong promoter sequences and high copy number of plasmidsthat can be utilized for production of the desired proteins in yeast.Yeast cells recognize leader sequences in cloned mammalian gene productsand secrete polypeptides bearing leader sequences (i.e., pre-peptides).

For long-term, high-yield production of a recombinant polypeptide,stable expression is preferred. For example, cell lines which stablyexpress the polypeptide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. A cell linesubstantially enriched in such cells can be then isolated to provide astable cell line.

A particularly preferred method of high-yield production of arecombinant polypeptide of the present invention is through the use ofdihydrofolate reductase (DHFR) amplification in DHFR-deficient CHOcells, by the use of successively increasing levels of methotrexate asdescribed in U.S. Pat. No. 4,889,803. The polypeptide obtained may be ina glycosylated form.

Antibodies disclosed herein can also be expressed in other eukaryoticcells, such as avian, fungal, insect, yeast, or plant cells. Thebaculovirus system provides an efficient means to introduce cloned genesinto insect cells. The materials for baculovirus/insect cell expressionsystems are commercially available in kit form from, inter alia,Invitrogen.

In addition to recombinant DNA technologies, the antibodies of thisinvention may be prepared by chemical synthesis technologies. Examplesof chemical synthesis technologies are solid phase synthesis and liquidphase synthesis. As a solid phase synthesis, for example, the amino acidcorresponding to the carboxy-terminus of the polypeptide to besynthesised is bound to a support which is insoluble in organic solventsand, by alternate repetition of reactions (e.g., by sequentialcondensation of amino acids with their amino groups and side chainfunctional groups protected with appropriate protective groups), thepolypeptide chain is extended. Solid phase synthesis methods are largelyclassified by the tBoc method and the Fmoc method, depending on the typeof protective group used. Totally synthetic proteins are disclosed inthe literature (Brown A et al., 1996).

The antibodies of the present invention can be produced, formulated,administered, or generically used in other alternative forms that can bepreferred according to the desired method of use and/or production. Theproteins of the invention can be post-translationally modified, forexample by glycosylation. The polypeptides or proteins of the inventioncan be provided in isolated (or purified) biologically active form, oras precursors, derivatives and/or salts thereof.

In another embodiment, IL-31 binding molecules or IL-31 antagonistsfusion proteins can be used for in vivo killing of target tissues wherethe IL-31 receptors are expressed) (See, generally, Hornick et al.,Blood 89:4437-47, 1997). The described fusion proteins enable targetingof the IL-31 binding molecules or IL-31 antagonists a desired site ofaction, thereby providing an elevated local concentration of IL-31binding molecules or IL-31 antagonists, targeting an undesirable cell ortissue (i.e., a tumor or a leukemia), and the fused cytokine mediatedimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

The IL-31 binding molecules or IL-31 antagonists of the presentinvention can be measured for their ability to inhibit, block, orneutralize the IL-31 ligand as determined by various in vivo modelsknown in the art and described herein, including but not limited to theNC/Nga model, the Ova epicutaneous model, the chronic hypersensitivitymodel, and the chronic hapten model.

Both skin-homing T cells and epidermal keratinocytes have beenimplicated in the pathology of skin diseases in humans. IL-31 mRNA andprotein expression is restricted to the skin-homing CLA+ T cellpopulation in humans. See U.S. patent application Ser. No. 11/353,427,filed Feb. 14, 2006, incorporated herein by reference. As such, anantagonist to IL-31, including an antibody or receptor antagonist willbe useful in treating skin and epidermal diseases which have expressionof CLA+ T cells. Such diseases include, for example, atopic dermatitis,contact dermatitis, drug-induced allergic reactions, skin-tropic virusesand viral associated pruritis, vitiligo, cutaneous T cell lymphoma,alopecia aerata, acne rosacea, acne vulgaris, prurigo nodularis, andbullous pemphigoid. Chemokine markers such as TARC and MDC are useful tomeasure the effect of a neutralizing monoclonal antibody to IL-31. Theinhibitory effects of treatment with IL-31 binding molecules or IL-31antagonists described herein can be measured by monitoring the levels ofTARC and MDC.

Contact Dermatitis

Allergic contact dermatitis is defined as a T cell mediated immunereaction to an antigen that comes into contact with the skin. The CLA+ Tcell population is considered to be involved in the initiation ofdermatitis since allergen dependent T cell responses are largelyconfined to the CLA+ population of cells (See Santamaria-Babi, L. F., etal., J Exp Med: 181, 1935, (1995)). Recent data has found that onlymemory (CD45RO+) CD4+ CLA+ and not CD8+ T cells proliferate and produceboth type-1 (IFN-) and type-2 (IL-5) cytokines in response to nickel, acommon contact hypersensitivity allergen. Furthermore, cells expressingCLA in combination with CD4, CD45RO (memory) or CD69 are increased afternickel-specific stimulation and express the chemokine receptors CXCR3,CCR4, CCR10 but not CCR6. See Moed H., et al., Br J Dermatol: 51, 32,(2004).

In animal models, it has been demonstrated that allergic contactdermatitis is T-cell dependent and that the allergic-responsive T cellsmigrate to the site of allergen application. See generally: Engeman T.M., et al., J Immunol: 164, 5207, (2000); Ferguson T. A. & Kupper T. S.J Immunol: 150, 1172, (1993); and Gorbachev A. V. & Fairchild R. L. CritRev Immunol: 21, 451(2001). Since CLA+ T cells produce IL-31 and IL-31stimulation of skin keratinocytes can induce pro-inflammatorychemokines, such as TARC and MDC, IL-31 may be involved in thepathophysiology of contact dermatitis. By using a neutralizing IL-31antibody in a mouse model of contact hypersensitivity. See Example 5.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofContact Hypersensitivity by inhibition, reduction, neutralization,prevention or blocking the inflammation and/or scratching associatedwith disease.

Atopic Dermatitis

Atopic dermatitis (AD) is a chronically relapsing inflammatory skindisease with a dramatically increasing incidence over the last decades.Clinically AD is characterized by highly pruritic often excoriatedplaques and papules that show a chronic relapsing course. The diagnosisof AD is mostly based on major and minor clinical findings. See HanifinJ. M., Arch Dermatol: 135, 1551 (1999). Histopathology revealsspongiosis, hyper and focal parakeratosis in acute lesions, whereasmarked epidermal hyperplasia with hyper and parakeratosis,acanthosis/hypergranulosis and perivascular infiltration of the dermiswith lymphocytes and abundant mast cells are the hallmarks of chromiclesions.

T cells play a central role in the initiation of local immune responsesin tissues and evidence suggests that skin-infiltrating T cells inparticular, may play a key role in the initiation and maintenance ofdisregulated immune responses in the skin. Approximately 90% ofinfiltrating T cells in cutaneous inflammatory sites express thecutaneous lymphocyte-associated Ag (CLA+) which binds E-selectin, aninducible adhesion molecule on endothelium (reviewed in Santamaria-BabiL. F., et al., Eur J Dermatol: 14, 13, (2004)). A significant increasein circulating CLA+ T cells among AD patients compared with controlindividuals has been documented (See Teraki Y., et al., Br J Dermatol:143, 373 (2000), while others have demonstrated that memory CLA+ T cellsfrom AD patients preferentially respond to allergen extract compared tothe CLA− population (See Santamaria-Babi, L. F., et al., J Exp Med: 181,1935, (1995)). In humans, the pathogenesis of atopic disorders of theskin have been associated with increases in CLA+ T cells that expressincreased levels of Th-2-type cytokines like IL-5 and IL-13 9, 10. SeeAkdis M., et al., Eur J Immunol: 30, 3533 (2000); and Hamid Q., et al.,J Allergy Clin Immunol: 98, 225 (1996).

NC/Nga Mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histophathologyand immunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139(2003). Therefore, the development of AD in NC/Nga is a useful model forthe evaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel J.M., et al., J Clin Invest, 101: 1614, (1998). This protocol can bemodified in order to induce skin barrier disregulation and pruritis bysensitizing DO11.10 OVA TCR transgenic mice with OVA. Increasing thenumber of antigen-specific T cells that could recognize the sensitizingantigen may increase the level of inflammation in the skin to inducevisible scratching behaviour and lichenification/scaling of the skin.

Both the NC/Nga spontaneous AD model and the OVA epicutaneous DO11.10model are used to evaluate the ability of the IL-31 binding molecules orIL-31 antagonists described herein to inhibit, reduce, or neutralize theeffects of IL-31. Administration of IL-31 binding molecules or IL-31antagonists can result in a reduction in scratching that can beeffective in treating pruritic diseases including, but not limited to,atopic dermatitis, prurigo nodularis, and eczema, since cessation ofscratching will stop progression of dermatitis, the development of whichis dependent on scratching.

Additional models to measure the inhibitory effects of the IL-31 bindingmolecules or IL-31 antagonists described herein are described byUmeuchi, H. et al., European Journal of Pharmacology, 518: 133-139,2005; and by Yoo, J. et al., J. Experimental Medicine, 202:541-549,2005.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofdermatitis and pruritic diseases including atopic dermatitis, prurigonodularis, and eczema by inhibition, reduction, prevention or blockingthe inflammation and/or scratching associated with disease.

Drug-Induced Delayed Type Cutaneous Allergic Reactions

Drug-induced delayed type cutaneous allergic reactions are veryheterogeneous and may mirror many distinct pathophysiological events.See Brockow K., et al., Allergy: 57, 45 (2002). Immunological mechanismsinvolved in these reactions have been shown as either antibody or cellmediated. In immediate drug allergy an IgE-mediated antibody reactioncan be demonstrated by a positive skin prick and/or intradermal testafter 20 min, whereas non-immediate reactions to drugs can occur morethan one hour after last drug intake and are often T-cell mediated.Non-immediate T-cell mediated delayed type reactions can occur inpatients with adverse drug reactions to penicillins for example.Proliferative T cell responses to penicillins have been shown to berestricted to the memory (CD45RO+) CLA+ subpopulation of T cells frompenicillin allergic patients whereas the CD45RO+ CLA− subset shows noproliferative response. See Blanca M., Leyva L., et al., Blood Cells MolDis: 31, 75 (2003). Delayed-type hypersensitivity (DTH) reactions can beartificially reproduced in mice, allowing assessment of factors that maybe involved in the initiation and perpetuation of the DTH response.11-31 neutralizing IL-31 binding molecules or IL-31 antagonists could beeffective in delayed type hypersensitivity reactions.

Toxic epidermal necrolysis (TEN) is a very rare but extremely severedrug reaction characterized by widespread apoptosis of epidermis withextensive blisters. Studies have shown that lymphocytes infiltrating theblister are CLA+ T cells and can exhibit cytotoxicity towards epidermalkeratinocytes. See Leyva L., et al., J Allergy Clin Immunol: 105, 157(2000); and Nassif A., Bensussan A., et al., J Allergy Clin Immunol:114, 1209 2004). A transgenic mouse system, whereby OVA is expressedunder the control of the keratin-5 (K5) promoter in the epidermal andhair follicular keratinocytes of mice, has been generated to establishan animal model for TEN. OVA specific CD8+ T cells, when adoptivelytransferred into K5-OVA mice, undergo activation and proliferation inthe skin-draining lymph nodes and target the skin of K5-OVA mice,resulting in development of skin lesions that are reminiscent of TEN.See Azukizawa H., et al., Eur J Immunol: 33, 1879 (2003).

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofTEN by inhibition, reduction, prevention or blocking the inflammationand/or scratching associated with disease.

Bullous Pemphigoid

Bullous pemphigoid is a subepidermal disorder which manifests assubepidermal blisters with a dermal infiltrate of neutrophils andeosinophils. Diagnosis is characterized by the presence ofantigen-specific antibodies against specific adhesion proteins of theepidermis and dermal-epidermal junction. See Jordon R. E., et al., JAMA:200, 751 (1967). Studies analyzing the role of T cells in thepathogenesis of bullous pemphigoid by analysis of PBL and skin blister Tcells have found a predominance of CLA+ T cells expressing increasedlevels of Th2-cytokines like IL-4 and IL-13. See Teraki Y., et al., JInvest Dermatol: 117, 1097 (2001). In bullous pemphigoid patientsfollowing therapy with systemic corticosteroids, the frequency of CLA+,but not CLA−, interleukin-13-producing cells is significantly decreased.Decreases in CLA+ cells following corticosteroid treatment is associatedwith clinical improvement. See Teraki, ibid.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofbullous pemphigoid by inhibition, reduction, prevention or blocking theinflammation and/or scratching associated with disease.

Alopecia Areata

Alopecia areata (AA) is regarded as a tissue-restricted autoimmunedisease of hair follicles in which follicular activity is arrestedbecause of the continued activity of lymphocytic infiltrates. AA resultsin patches of complete hair loss anywhere on the body, though actualloss of hair follicles does not occur, even in hairless lesions.Although clinical signs of inflammation are absent, skin biopsies fromsites of active disease show perifollicular lymphocytic inflammation ofprimarily CD4+ cells, along with a CD8+ intrafollicular infiltrate. SeeKalish R. S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164 (2003).

Studies have shown that scalp skin infiltrating CD4+ or CD8+ lymphocytesexpress CLA and, in peripheral blood of individuals with AA, the percentof CLA+ CD4+ or CD8+ lymphocytes is significantly higher than that ofnormal controls. Furthermore, patients with severe or progressive AAshow a much higher CLA-positivity compared to patients recovering fromthe disease and a decrease in percent CLA+ cells parallels a goodclinical course. See Yano S., et al., Acta Derm Venereol: 82, 82 (2002).These studies therefore suggest that CLA+ lymphocytes may play animportant role in AA. Xenograft models have demonstrated that activatedT cells are likely to play a role in the pathogenesis of AA. Lesionalscalp from AA patients grafted onto nude mice regrows hair coincidentwith a loss of infiltrating lymphocytes from the graft and, transfer ofactivated lesional T cells to SCID mice can transfer hair loss to humanscalp explants on SCID mice. See Kalish R. S. & Gilhar A. J InvestigDermatol Symp Proc: 8, 164 (2003).

A variety of immunomodulating therapies are part of the usual treatmentfor this disorder however none of these treatments have been consistentin their efficacy. See Tang L., et al., J Invest Dermatol: 120, 400(2003); Tang L., et al., (2004); and Tang L., et al., J Am AcadDermatol: 49, 1013 (2003). Nevertheless, their uses in valid animalmodels provide a tool to dissect out molecular mechanisms of therapeuticeffects. See Shapiro J., et al., J Investig Dermatol Symp Proc: 4, 239(1999); Tang L., et al., Old wine in new bottles: reviving old therapiesfor alopecia areata using rodent models (2003); and Verma D. D., et al.,Eur J Dermatol: 14, 332 (2004).

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofalopecia areata by inhibition, reduction, prevention or blocking theinflammation and/or scratching associated with disease.

Acne Rosacea/Acne vulgaris

Acne vulgaris, a disorder of the pilosebaceous apparatus, is the mostcommon skin problem of adolescence. Abnormalities in follicularkeratinization are thought to produce the acne lesion. Acne rosacea isdifferentiated from acne vulagaris by the presence of red papules,pustules, cysts and extensive telangiectasias, but the absence ofcomedones (white heads). Increased sebum excretion from sebaceous glandsis a major factor in the pathophysiology of acne vulgaris. Othersebaceous gland functions are also associated with the development ofacne, including sebaceous proinflammatory lipids; different cytokinesproduced locally; periglandular peptides and neuropeptides, such ascorticotrophin-releasing hormone, which is produced by sebocytes; andsubstance P, which is expressed in the nerve endings at the vicinity ofhealthy-looking glands of acne patients. See Zouboulis C. C. ClinDermatol: 22, 360 (2004).

Although the pathophysiology of acne vulgaris and acne rosacea remainsunknown, clinical observations and histopathologic studies suggest thatinflammation of the pilosebaceous follicle may be central to thepathogenesis of rosacea and acne vulgaris. Early studies on analysis ofT cell subsets infiltrating rosacea legions indicated that the majorityof T cells expressed CD4. See Rufli T. & Buchner S. A. Dermatologica:169, 1 (1984).

CD4+ T cells produce IL-31 and IHC analysis of skin for IL-31 expressionsuggests that IL-31 is expressed in sebaceous and sweat glands. IL-31stimulation of epidermal keratinocytes induces expression of chemokineswhich likely results in cellular infiltration suggesting that IL-31 maycontribute to the pro-inflammatory response in skin. See Dillon S. R.,et al., Nat Immunol: 5, 752 (2004). IL-31 may therefore contribute tothe pathophysiology of acne rosacea and acne vulgaris.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofacne vulgaris by inhibition, reduction, prevention or blocking theinflammation and/or scratching associated with disease.

Prurigo Nodularis

Prurigo nodularis is an eruption of lichenified or excoriated nodulescaused by intractable pruritus that is difficult to treat. While chronicrubbing results in lichenification, and scratching in linearexcoriations, individuals who pick and gouge at their itchy, irritatedskin tend to produce markedly thickened papules known as prurigonodules. Although prurigo nodularis is not specific to atopicdermatitis, many patients with these nodules also have an atopicreaction, which manifests as allergic rhinitis, asthma, or food allergy.T cells represent the majority of infiltrating cells in prurigo lesionsand these lesions often represents the most pruritic skin lesion inatopy patients.

Topical treatment of prurigo nodularis with capsaicin, an anti-pruriticalkaloid that interferes with the perception of prurities and pain bydepletion of neuropeptides like substance P in small sensory cutaneousnerves, has proven to be an effective and safe regimen resulting inclearing of the skin lesions. See Stander S., et al., J Am AcadDermatol: 44, 471 (2001). Studies of the itch response in NC/Nga miceusing capsaicin treatment showed that the spontaneous development ofdermatitis lesions was almost completely prevented. Furthermore, theelevation of serum IgE levels was significantly suppressed andinfiltrating eosinophils and mast cell numbers in lesional skin ofcapsaicin treated mice were reduced. See Mihara K., et al., Br JDermatol: 151, 335 (2004). The observations from this group suggest thatscratching behaviour might contribute to the development of dermatitisby enhancing various immunological responses, therefore implying thatprevention of the itch sensation and/or itch-associated scratchingbehaviour might be an effective treatment for AD. See Mihara K., et al.,Br J Dermatol: 151, 335 (2004). Thus, the anti-Il-31 antibodiesdescribed herein will be useful in minimizing the effects of AD, prurigonodularis, and other pruritic diseases as they are shown herein toreduce the amount of scratching in NC/Nga mice.

Chronic delivery of IL-31 induces pruritis and alopecia in mice followedby the development of skin lesions resembling dermatitis suggesting thatIL-31 may induce itching. See Dillon S. R., et al., Nat Immunol: 5, 752(2004). The involvement of IL-31 in induction of the itch response bytwo methods (i) capsaicin treatment of IL-31-treated mice and (ii) IL-31treatment of Tac1 knockout mice, which have significantly reducednociceptive pain responses because of lack of expression ofneuropeptides is tested in Example 10. In addition, whetherneutralization of IL-31 in IL-31 treated mice with IL-31 bindingmolecules or IL-31 antagonists could prevent pruritis and alopecia istested in Example 12.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofprurigo nodularis by inhibition, reduction, prevention or blocking theinflammation and/or scratching associated with disease.

Skin-Tropic Viruses and Viral Associated Pruritis

Herpes Simplex Virus (HSV)-specific CD8+ T cells in the peripheral bloodand HSV-specific CD8+ T cells recovered from herpes lesions express highlevels of CLA where as non-skin-tropic herpes virus-specific CD8+ Tcells lack CLA expression. See Koelle D. M., et al., J Clin Invest: 110,537 (2002). HSV-2 reactive CD4+ T lymphocytes also express CLA, but atlevels lower than those previously observed for CD8+ T lymphocytes. SeeGonzalez J. C., et al., J Infect Dis: 191, 243 (2005). Pruritis has alsobeen associated with herpes viral infections (See Hung K. Y., et al.,Blood Purif: 16, 147 (1998). though other viral diseases, like HIV, havealso been associated with pruritic skin lesions. Severe, intractablepruritus, often associated with erythematopapular skin lesions andhypereosinophilia, is a condition observed in some nonatopic,HIV-infected patients 36. See Singh F. & Rudikoff D, Am J Clin Dermatol;4, 177 (2003); and Milazzo F., Piconi S., et al., Allergy: 54, 266(1999).

The association of skin-tropic viruses with pruritis and CLA+ T cellssuggests that IL-31 producing T cells may be involved in thepathophysiology of viral infections.

Thus, neutralization of IL-31 by the IL-31 binding molecules or IL-31antagonists described herein may be used to improve clinical outcome ofpruritis associated with skin-tropic viruses by inhibition, reduction,prevention or blocking the inflammation and/or scratching associatedwith disease.

IL-31 involvement in the induction of itch response, and its reduction,blocking, inhibition or neutralization by the IL-31 binding moleculesdescribed herein, can be measured in a number of ways.

Method I (Capsaicin Treatment of IL-31 Treated Mice):

Ten week old BALB/c animals (CRL) are anaesthetized and injected with along-lasting analgesic agent, bupranorphine hydrochloride,subcutaneously at 0.1 mg/kg before injection of 0.25 ml of 4 mg/mlsolution of capsaicin in 10% ethanol+10% Tween-80 in salinesubcutaneously into scruff of neck. Animals are kept anaesthetized forat least 30 min following neurotoxin treatment. Forty-eight hours later,14-day osmotic pumps are implanted subcutaneously for continuousdelivery of 20 ug/day of IL-31 for 14 days. Mice are monitored daily for6 days for alopecia and pruritis using the following criteria: 0=noscratching, animal appears normal, 1=thinning of coat in small areas,scratching noted, 2=minor hair loss (small patches), scratching,3=moderate hair loss, scratching, and 4=severe hair loss, excessivescratching. When this experiment was performed the results demonstratedthat while non-capsaicin-treated mice showed a mean scratch/hairlossscore of 2.625 following three days of IL-31 delivery, capsaicin-treatedmice showed a significantly lower score of 1. Thus mice treated withcapsaicin prior to IL-31 delivery showed both a delay in incidence ofscratching and hairloss and a lower score in the intensity of scratchingand hairloss over the six days of the experiment. These data suggestthat IL-31 does induce some neuronal component that contributes to thealopecia and pruritis induced by IL-31. Therefore, neutralization ofIL-31 by IL-31 binding molecules or IL-31 antagonists may decrease theincidence and intensity of itch, and therefore dermatitis, in patientssuffering from skin disorders that involve itch.

Method II:

Mice that are homozygous null for the Tac1 gene express no detectiblesubstance P or neurokinin A. These mice have significantly reducednociceptive pain responses to moderate to intense stimuli and aretherefore a useful tool for studying the contribution of tachykininpeptides to pain/itch processing and inflammatory disease states. Twelveweek old, Tac1 knockout mice were implanted with 14-day osmotic pumpsdelivering 1 ug/day of IL-31 protein and observed daily for alopecia andpruritis using the following criteria: 0=no scratching, animal appearsnormal, 1=thinning of coat in small areas, scratching noted, 2=minorhair loss (small patches), scratching, 3=moderate hair loss, scratching,and 4=severe hair loss, excessive scratching.

Results of this study show that Tac1 deficient mice were lesssusceptible to IL-31 induced scratching/hairloss compared to wildtypecontrol mice. While 100% (10/10) of wildtype mice had developed evidenceof scratching and hairloss by day 6 of IL-31 treatment, only 33.3% (2/6)Tac1 deficient mice were showing signs of scratching and hairloss at thesame time-point. These data show that IL-31 induces a neuronal componentthat contributes to the scratch/hairloss phenotype in IL-31-treated miceand neutralization of IL-31 by IL-31 binding molecules or IL-31antagonists may decrease the incidence and intensity of scratching inthe context of dermatitis.

Methods III (Administration of IL-31 Neutralizing Antibody):

Normal female BALB/c mice (CRL) approximately 8 to 12 weeks old wereimplanted subcutaneously with 14-day osmotic pumps (Alzet, #2002)delivering 1 ug/day mIL-31. Groups of mice received intraperitoneal(i.p.) injections of rat anti-mouse IL-31 monoclonal antibody 10 mg/kg(200 ug/mouse) twice weekly starting 1 week prior to IL-31 delivery.Control groups of mice received i.p. injections of vehicle (PBS/0.1%BSA) with the identical dosing schedules. Mice were scored daily foralopecia and pruritis using the following criteria: 0=no scratching,animal appears normal, 1=thinning of coat in small areas, scratchingnoted, 2=minor hair loss (small patches), scratching, 3=moderate hairloss, scratching, and 4=severe hair loss, excessive scratching.

In all experiments, mice treated with rat anti-mIL-31 mAb had a delay inonset of symptoms of approximately 5 to 7 days and a lower overall scorefor alopecia and pruritis. All groups of mAb treated mice (regardless ofdose frequency or concentration) developed alopecia and pruritis similarto control mice by 13 day of the study. These data suggest thatneutralization of IL-31 by IL-31 binding molecules or IL-31 antagonistscan delay the onset of the scratch/hairloss response induced by IL-31.The effects of IL-31 binding molecules or IL-31 antagonists are measuredby inhibition of scratching, itching, dermatitis, a reduction in IL-31RAexpression in kerotinocytes, and/or a reduction in score for alopeciaand pruritis.

Inflammation is a protective response by an organism to fend off aninvading agent. Inflammation is a cascading event that involves manycellular and humoral mediators. On one hand, suppression of inflammatoryresponses can leave a host immunocompromised; however, if leftunchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease and the like), septic shock andmultiple organ failure. Importantly, these diverse disease states sharecommon inflammatory mediators. The collective diseases that arecharacterized by inflammation have a large impact on human morbidity andmortality. Therefore it is clear that anti-inflammatory antibodies andbinding polypeptides, such as anti-IL-31 antibodies and bindingpolypeptides described herein, could have crucial therapeutic potentialfor a vast number of human and animal diseases, from asthma and allergyto autoimmunity and septic shock. As such, use of anti-inflammatory antiIL-31 antibodies and binding polypeptides described herein can be usedtherapeutically as IL-31 antagonists described herein, particularly indiseases such as arthritis, endotoxemia, inflammatory bowel disease,psoriasis, related disease and the like.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory antibodies and binding polypeptides, such asanti-IL-31 antibodies and binding polypeptides of the present invention.For Example, rheumatoid arthritis (RA) is a systemic disease thataffects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002). Oneof those mediators could be IL-31, and as such a molecule that binds orinhibits IL-31, such as anti IL-31 antibodies or binding partners, couldserve as a valuable therapeutic to reduce inflammation in rheumatoidarthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol 92:8955-959, 1995).

The administration of soluble IL-31RA comprising polypeptides (includingheterodimeric and multimeric receptors described herein), such asIL-31RA-Fc4 or other IL-31RA soluble and fusion proteins to these CIAmodel mice can be used to evaluate the use of IL-31RA to amelioratesymptoms and alter the course of disease. As a molecule that modulatesimmune and inflammatory response, IL-31, may induce production of SAA,which is implicated in the pathogenesis of rheumatoid arthritis, IL-31binding molecules or IL-31 antagonists may reduce SAA activity in vitroand in vivo, the systemic or local administration of IL-31 bindingmolecules or IL-31 antagonists can potentially suppress the inflammatoryresponse in RA.

2. Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory antibodies and binding polypeptides, such asanti-IL-31 antibodies and binding polypeptides of the present invention,could aid in preventing and treating endotoxemia in humans and animals.Other potential therapeutics include IL-31RA polypeptides, solubleheterodimeric and multimeric receptor polypeptides, or anti IL-31antibodies or binding partners of the present invention, and the like,could serve as a valuable therapeutic to reduce inflammation andpathological effects in endotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals. Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that 1 ug injection of E. coli LPS into a C57Bl/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, IL-10,and G-CSF. Since LPS induces the production of pro-inflammatory factorspossibly contributing to the pathology of endotoxemia, theneutralization of IL-31 activity, SAA or other pro-inflammatory factorsby antagonizing IL-31 polypeptide can be used to reduce the symptoms ofendotoxemia, such as seen in endotoxic shock. Other potentialtherapeutics include IL-31 binding molecules or IL-31 antagonists.

3 Inflammatory Bowel Disease. IBD

In the United States approximately many people suffer from InflammatoryBowel Disease (IBD) which can affect either colon and rectum (Ulcerativecolitis) or both, small and large intestine (Crohn's Disease). Thepathogenesis of these diseases is unclear, but they involve chronicinflammation of the affected tissues. Potential therapeutics includeIL-31RA polypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti-IL-31 antibodies or binding partners of thepresent invention, and the like, could serve as a valuable therapeuticto reduce inflammation and pathological effects in IBD and relateddiseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining Treatments including corticosteroids immunosuppressives (e.g.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of anti-IL-31 antibodies or binding partners, solubleIL-31RA comprising polypeptides (including heterodimeric and multimericreceptors), such as IL-31RA-Fc4 or other IL-31RA soluble and fusionproteins to these TNBS or DSS models can be used to evaluate the use ofIL-31 antagonists to ameliorate symptoms and alter the course ofgastrointestinal disease. IL-31 may play a role in the inflammatoryresponse in colitis, and the neutralization of IL-31 activity byadministrating IL-31 binding molecules or IL-31 antagonists is apotential therapeutic approach for IBD.

4. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. IL-31RApolypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti-IL-31 antibodies or binding partners of thepresent invention, and the like, could serve as a valuable therapeuticto reduce inflammation and pathological effects in psoriasis, otherinflammatory skin diseases, skin and mucosal allergies, and relateddiseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases r

IL-31 was isolated from tissue known to have important immunologicalfunction and which contain cells that play a role in the immune system.IL-31 is expressed in CD3+ selected, activated peripheral blood cells,and it has been shown that IL-31 expression increases after T cellactivation. Moreover, results of experiments described in the Examplessection herein suggest that polypeptides of the present invention canhave an effect on the growth/expansion of monocytes/macrophages,T-cells, B-cells, NK cells and/or differentiated state ofmonocytes/macrophages, T-cells, B-cells, NK cells or these cells'progenitors. Factors that both stimulate proliferation of hematopoieticprogenitors and activate mature cells are generally known, however,proliferation and activation can also require additional growth factors.For example, it has been shown that IL-7 and Steel Factor (c-kit ligand)were required for colony formation of NK progenitors. IL-15+IL-2 incombination with IL-7 and Steel Factor was more effective (Mrózek etal., Blood 87:2632-2640, 1996). However, unidentified cytokines may benecessary for proliferation of specific subsets of NK cells and/or NKprogenitors (Robertson et. al., Blood 76:2451-2438, 1990). Similarly,IL-31 may act alone or in concert or synergy with other cytokines toenhance growth, proliferation expansion and modification ofdifferentiation of monocytes/macrophages, T-cells, B-cells or NK cells.

The present invention provides a method for inhibiting activation ordifferentiation of monocytes/macrophages. Monocytes are incompletelydifferentiated cells that migrate to various tissues where they matureand become macrophages. Macrophages play a central role in the immuneresponse by presenting antigen to lymphocytes and play a supportive roleas accessory cells to lymphocytes by secreting numerous cytokines.Macrophages can internalize extracellular molecules and upon activationhave an increased ability to kill intracellular microorganisms and tumorcells. Activated macrophages are also involved in stimulating acute orlocal inflammation.

The tissue distribution of receptors for a given cytokine offers astrong indication of the potential sites of action of that cytokine.Expression of IL-31RA was seen in monocytes and B-cells, with a dramaticincrease of expression upon activation for CD3+, CD4+, and CD8+ T-cells.In addition, two monocytic cell lines, THP-1 (Tsuchiya et al., Int. J.Cancer 26:171-176, 1980) and U937 (Sundstrom et al., Int. J. Cancer17:565-577, 1976), were also positive for IL-31RA expression.

Expression of OSMR is reported to be very broad (Mosley et al, JBC271:32635-32643, 1996). This distribution of IL-31RA and OSM receptorssupports a role for IL-31 in immune responses, especially expansion ofT-cells upon activation or a role in the monocyte/macrophage arm of theimmune system.

Thus, particular embodiments of the present invention are directedtoward use of IL-31 binding molecules or IL-31 antagonists asantagonists in inflammatory and immune diseases or conditions such aspancreatitis, type I diabetes (IDDM), pancreatic cancer, pancreatitis,Graves Disease, inflammatory bowel disease (IBD), Crohn's Disease, colonand intestinal cancer, diverticulosis, autoimmune disease, sepsis, organor bone marrow transplant; inflammation due to trauma, surgery orinfection; amyloidosis; splenomegaly; graft versus host disease; andwhere inhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells, CD4+ and CD8+ cells),suppression of immune response to a pathogen or antigen. Moreover thepresence of IL-31RA expression in activated immune cells such asactivated CD4+ and CD19+ cells showed that IL-31RA receptor may beinvolved in the body's immune defensive reactions against foreigninvaders: such as microorganisms and cell debris, and could play a rolein immune responses during inflammation and cancer formation. As such,antibodies and binding partners of the present invention that areagonistic or antagonistic to IL-31RA receptor function, such as IL-31,can be used to modify immune response and inflammation.

IL-31 binding molecules or IL-31 antagonists may also be used withindiagnostic systems for the detection of circulating levels of IL-31.Within a related embodiment, antibodies or other agents thatspecifically bind to IL-31 polypeptides can be used to detectcirculating IL-31 polypeptides. Elevated or depressed levels of ligandpolypeptides may be indicative of pathological conditions, includingcancer. IL-31 polypeptides may contribute to pathologic processes andcan be an indirect marker of an underlying disease.

In atherosclerotic lesions, one of the first abnormalities islocalization of monocyte/macrophages to endothelial cells. These lesionscould be prevented by use of antagonists to IL-31. IL-31 bindingmolecules or IL-31 antagonists can also be used as antagonists to theIL-31. Moreover, monoblastic leukemia is associated with a variety ofclinical abnormalities that reflect the release of the biologic productsof the macrophage, examples include high levels of lysozyme in the serumand urine and high fevers. Moreover, such leukemias exhibit an abnormalincrease of monocytic cells. These effects could possibly be preventedby antagonists to IL-31, such as described herein. Moreover, anti-IL-31can be conjugated to molecules such as toxic moieties and cytokines, asdescribed herein to direct the killing of leukemia monocytic cells.

As IL-31 is expressed in a T-cell, macrophage and monocyte-specificmanner, and these diseases involve abnormalities in monocytic cells,such as cell proliferation, function, localization, and activation, thepolynucleotides, polypeptides, and antibodies of the present inventioncan be used to as diagnostics to detect such monocytic cellabnormalities, and indicate the presence of disease. Such methodsinvolve taking a biological sample from a patient, such as blood,saliva, or biopsy, and comparing it to a normal control sample.Histological, cytological, flow cytometric, biochemical and othermethods can be used to determine the relative levels or localization ofIL-31, or cells expressing IL-31, i.e., monocytes, in the patient samplecompared to the normal control. A change in the level (increase ordecrease) of IL-31 expression, or a change in number or localization ofmonocytes (e.g., increase or infiltration of monocytic cells in tissueswhere they are not normally present) compared to a control would beindicative of disease. Such diagnostic methods can also include usingradiometric, fluorescent, and colorimetric tags attached topolynucleotides, polypeptides or antibodies of the present invention.Such methods are well known in the art and disclosed herein.

IL-31 has been shown to be expressed in activated mononuclear cells, andmay be involved in regulating inflammation. As such, polypeptides of thepresent invention can be assayed and used for their ability to modifyinflammation, or can be used as a marker for inflammation. Methods todetermine proinflammatory and antiinflammatory qualities of IL-31 areknown in the art and discussed herein. Moreover, it may be involved inup-regulating the production of acute phase reactants, such as serumamyloid A (SAA), α1-antichymotrypsin, and haptoglobin, and thatexpression of IL-31RA receptor ligand may be increased upon injection oflipopolysaccharide (LPS) in vivo that are involved in inflammatoryresponse (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,2000). Production of acute phase proteins, such as SAA, is considered sshort-term survival mechanism where inflammation is beneficial; however,maintenance of acute phase proteins for longer periods contributes tochronic inflammation and can be harmful to human health. For review, seeUhlar, C M and Whitehead, A S, Eur. J. Biochem. 265:501-523, 1999, andBaumann H. and Gauldie, J. Immunology Today 15:74-80, 1994. Moreover,the acute phase protein SAA is implicated in the pathogenesis of severalchronic inflammatory diseases, is implicated in atherosclerosis andrheumatoid arthritis, and is the precursor to the amyloid A proteindeposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus, wherea ligand such as IL-31 that acts as a pro-inflammatory molecule andinduces production of SAA, antagonists would be useful in treatinginflammatory disease and other diseases associated with acute phaseresponse proteins induced by the ligand. Such antagonists are providedby the present invention. For example, a method of reducing inflammationcomprises administering to a mammal with inflammation an amount of acomposition of IL-31, or anti-IL-31 antibody (e.g., neutralizingantibody) that is sufficient to reduce inflammation. Moreover, a methodof suppressing an inflammatory response in a mammal with inflammationcan comprise: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising a IL-31 polypeptide or anti-IL-31antibody as described herein in an acceptable pharmaceutical carrier;(3) determining a post administration level of serum amyloid A protein;(4) comparing the level of serum amyloid A protein in step (1) to thelevel of serum amyloid A protein in step (3), wherein a lack of increaseor a decrease in serum amyloid A protein level is indicative ofsuppressing an inflammatory response.

Tissue distribution of the mRNA corresponding it's IL-31RA receptor cDNAshowed that mRNA level was highest in monocytes and prostate cells, andis elevated in activated monocytes, and activated CD4+, activated CD8+,and activated CD3+ cells. Hence, IL-31RA receptor is also implicated ininducing inflammatory and immune response. Thus, particular embodimentsof the present invention are directed toward use of IL-31 bindingmolecules or IL-31 antagonists in inflammatory and immune diseases orconditions such as, pancreatitis, type I diabetes (IDDM), pancreaticcancer, pancreatitis, Graves Disease, inflammatory bowel disease (IBD),Crohn's Disease, colon and intestinal cancer, diverticulosis, autoimmunedisease, sepsis, organ or bone marrow transplant; inflammation due totrauma, surgery or infection; amyloidosis; splenomegaly; graft versushost disease; and where inhibition of inflammation, immune suppression,reduction of proliferation of hematopoietic, immune, inflammatory orlymphoid cells, macrophages, T-cells (including Th1 and Th2 cells, CD4+and CD8+ cells), suppression of immune response to a pathogen orantigen. Moreover the presence of IL-31RA receptor and IL-31 expressionin activated immune cells such as activated CD3+, monocytes, CD4+ andCD19+ cells showed that IL-31RA receptor may be involved in the body'simmune defensive reactions against foreign invaders: such asmicroorganisms and cell debris, and could play a role in immuneresponses during inflammation and cancer formation. As such, IL-31 andIL-31-antibodies of the present invention that are agonistic orantagonistic to IL-31RA receptor function, can be used to modify immuneresponse and inflammation.

IL-31 binding molecules or IL-31 antagonists are useful to:)

1) Antagonize or block signaling via IL-31RA-comprising receptors in thetreatment of acute inflammation, inflammation as a result of trauma,tissue injury, surgery, sepsis or infection, and chronic inflammatorydiseases such as asthma, inflammatory bowel disease (IBD), chroniccolitis, splenomegaly, rheumatoid arthritis, recurrent acuteinflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response.

2) Antagonize or block signaling via the IL-31RA receptor receptors inthe treatment of autoimmune diseases such as IDDM, multiple sclerosis(MS), systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, and IBD to prevent or inhibit signaling in immune cells (e.g.lymphocytes, monocytes, leukocytes) via IL-31RA receptor (Hughes C etal., J. Immunol 153: 3319-3325, 1994). Alternatively antibodies, such asmonoclonal antibodies (MAb) to IL-31, can also be used as an antagonistto deplete unwanted immune cells to treat autoimmune disease. Asthma,allergy and other atopic disease may be treated with an MAb against, forexample, anti-IL-31 antibodies, soluble IL-31RA receptor solublereceptors or IL-31RA/CRF2-4 heterodimers, to inhibit the immune responseor to deplete offending cells. Blocking or inhibiting signaling viaIL-31RA, using the polypeptides and antibodies of the present invention,may also benefit diseases of the pancreas, kidney, pituitary andneuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinoma maybenefit. IL-31RA may serve as a target for MAb therapy of cancer wherean antagonizing MAb inhibits cancer growth and targets immune-mediatedkilling. (Holliger P, and Hoogenboom, H: Nature Biotech. 16: 1015-1016,1998). Mabs to soluble IL-31RA receptor monomers, homodimers,heterodimers and multimers may also be useful to treat nephropathiessuch as glomerulosclerosis, membranous neuropathy, amyloidosis (whichalso affects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

3) Agonize or initiate signaling via the IL-31RA receptors in thetreatment of autoimmune diseases such as IDDM, MS, SLE, myastheniagravis, rheumatoid arthritis, and IBD. IL-31 may signal lymphocytes orother immune cells to differentiate, alter proliferation, or changeproduction of cytokines or cell surface proteins that ameliorateautoimmunity Specifically, modulation of a T-helper cell response to analternate pattern of cytokine secretion may deviate an autoimmuneresponse to ameliorate disease (Smith J A et al., J. Immunol160:4841-4849, 1998). Similarly, IL-31 may be used to signal, depleteand deviate immune cells involved in asthma, allergy and atopoicdisease. Signaling via IL-31RA receptor may also benefit diseases of thepancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,pancreatitis, and pancreatic carcinoma may benefit. IL-31RA may serve asa target for MAb therapy of pancreatic cancer where a signaling MAbinhibits cancer growth and targets immune-mediated killing (Tutt, A L etal., J Immunol 161: 3175-3185, 1998). Similarly T-cell specificleukemias, lymphomas, plasma cell dyscrasia (e.g., multiple myeloma),and carcinoma may be treated with monoclonal antibodies (e.g.,neutralizing antibody) to IL-31RA-comprising soluble receptors of thepresent invention.

Generally, the dosage of administered IL-31 binding molecules or IL-31antagonists will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of IL-31 polypeptide which is in the range of from about 1 pg/kgto 10 mg/kg (amount of agent/body weight of patient), although a loweror higher dosage also may be administered as circumstances dictate. Oneskilled in the art can readily determine such dosages, and adjustmentsthereto, using methods known in the art.

Administration of a IL-31 binding molecules or IL-31 antagonists to asubject can be topical, inhalant, intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising IL-31 canbe prepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having IL-31 binding activity (Potts etal., Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising IL-31 binding molecules or IL-31antagonists having IL-31 binding activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having IL-31 binding activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having IL-31 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates at least aportion of the inflammatory response.

A pharmaceutical composition comprising IL-31 binding molecules or IL-31antagonists can be furnished in liquid form, in an aerosol, or in solidform. Liquid forms, are illustrated by injectable solutions, aerosols,droplets, topological solutions and oral suspensions. Exemplary solidforms include capsules, tablets, and controlled-release forms. Thelatter form is illustrated by miniosmotic pumps and implants (Bremer etal., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants in DrugDelivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.), pages95-123 (CRC Press 1995); Bremer et al., “Protein Delivery with InfusionPumps,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 239-254 (Plenum Press 1997); Yewey et al., “Delivery ofProteins from a Controlled Release Injectable Implant,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117(Plenum Press 1997)). Other solid forms include creams, pastes, othertopological applications, and the like.

The IL-31 binding molecules or IL-31 antagonists disclosed herein mayalso be formulated as immunoliposomes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Therapeutic formulations of the IL-31 binding molecules or IL-31antagonists are prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as Tween™, Pluronics™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to IL-31 in the one formulation. Alternatively, or in addition, thecomposition may comprise a chemotherapeutic agent or a cytokine. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Polypeptides having IL-31 binding activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim Biophys. Acta1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a IL-31 polypeptide or a IL-31antagonist (e.g., an antibody or antibody fragment that binds a IL-31polypeptide). Therapeutic polypeptides can be provided in the form of aninjectable solution for single or multiple doses, or as a sterile powderthat will be reconstituted before injection. Alternatively, such a kitcan include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of a therapeutic polypeptide.

Within an aspect the present invention pertains to an isolated antibodythat binds to human IL31, wherein said antibody is a humanized antibodyderived from the monoclonal antibody produced by the hybridoma depositedwith the American Type Culture Collection having the ATCC Patent DepositDesignation selected from: a) ATCC Patent Deposit Designation PTA-6815;b) ATCC Patent Deposit Designation PTA-6816; c) ATCC Patent DepositDesignation PTA-6829; d) ATCC Patent Deposit Designation PTA-6830; e)ATCC Patent Deposit Designation PTA-6831; f) ATCC Patent DepositDesignation PTA-6871; g) ATCC Patent Deposit Designation PTA-6872; h)ATCC Patent Deposit Designation PTA-6875; and i) ATCC Patent DepositDesignation PTA-6873; and wherein said antibody comprises a heavy chainimmunoglobulin constant domain which is a human IgG4. In an embodiment,the human IgG4 constant domain is a mutated form stable in solution andwith little or no complement activating activity. In a particularembodiment, the heavy chain immunoglobulin constant region domain is ahuman IgG4 constant domain with a Ser to Pro mutation at position 241(Kabat numbering).

Within another aspect these antibodies are used to treat diseasesincluding atopic dermatitis, contact dermatitis, drug-induced allergicreactions, skin-tropic viruses and viral associated pruritis, vitiligo,cutaneous T cell lymphoma, alopecia aerata, acne rosacea, acne vulgaris,prurigo nodularis, and bullous pemphigoid, as discussed herein.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein the immunoglobulin light chain constantregion domain is selected from the group consisting of the constantregion of a kappa or lambda human immunoglobulin light chain.Preferably, the immunoglobulin light chain constant region domain is theconstant region of a kappa human immunoglobulin light chain.

In an aspect, the present invention pertains to an isolated antibody asdisclosed herein wherein the heavy chain variable domain and the lightchain variable domain comprises the CDR sequences of clones 292.12.3.1,292.84.1.6, 292.63.5.3, 294.144.3.5, 292.39.5.3, 292.51.5.2,292.64.6.5.5, 292.105.4.1, 292.109.4.4, 292.118.6.4, and 292.72.3.1. TheCDR sequences are shown in the Figures and in Table 3, below.

In an aspect, the present invention pertains to an isolated antibody asdisclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 51, asecond CDR sequence consisting of SEQ ID NO: 52 or SEQ ID NO: 57, and athird CDR sequence consisting of SEQ ID NO:53; and b) the light chainvariable domain comprises first CDR sequence consisting of amino acidsequence SEQ ID NO: 54, a second CDR sequence consisting of SEQ ID NO:55, and a third CDR sequence consisting of SEQ ID NO:56. In anembodiment, the antibody is used to treat diseases including atopicdermatitis, contact dermatitis, drug-induced allergic reactions,skin-tropic viruses and viral associated pruritis, vitiligo, cutaneous Tcell lymphoma, alopecia aerata, acne rosacea, acne vulgaris, prurigonodularis, and bullous pemphigoid, as discussed herein. In anotherembodiment, chemokines such are TARC or MDC are measured.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 51, asecond CDR sequence consisting of SEQ ID NO: 58, and a third CDRsequence consisting of SEQ ID NO:59; and b) the light chain variabledomain comprises first CDR sequence consisting of amino acid sequenceSEQ ID NO: 60, a second CDR sequence consisting of SEQ ID NO: 61, and athird CDR sequence consisting of SEQ ID NO:62. In an embodiment, theantibody is used to treat diseases including atopic dermatitis, contactdermatitis, drug-induced allergic reactions, skin-tropic viruses andviral associated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis, and bullouspemphigoid, as discussed herein. In another embodiment, chemokines suchare TARC or MDC are measured.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 63, asecond CDR sequence consisting of SEQ ID NO: 64, and a third CDRsequence consisting of SEQ ID NO:65; and b) the light chain variabledomain comprises first CDR sequence consisting of amino acid sequenceSEQ ID NO: 66, a second CDR sequence consisting of SEQ ID NO: 67, and athird CDR sequence consisting of SEQ ID NO:68. In an embodiment, theantibody is used to treat diseases including atopic dermatitis, contactdermatitis, drug-induced allergic reactions, skin-tropic viruses andviral associated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis, and bullouspemphigoid, as discussed herein. In another embodiment, chemokines suchare TARC or MDC are measured.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 69, asecond CDR sequence consisting of SEQ ID NO: 70 or SEQ ID NO: 79, and athird CDR sequence consisting of SEQ ID NO:71; and b) the light chainvariable domain comprises first CDR sequence consisting of amino acidsequence SEQ ID NO: 72, a second CDR sequence consisting of SEQ ID NO:73, and a third CDR sequence consisting of SEQ ID NO:74. In anembodiment, the antibody is used to treat diseases including atopicdermatitis, contact dermatitis, drug-induced allergic reactions,skin-tropic viruses and viral associated pruritis, vitiligo, cutaneous Tcell lymphoma, alopecia aerata, acne rosacea, acne vulgaris, prurigonodularis, and bullous pemphigoid, as discussed herein. In anotherembodiment, chemokines such are TARC or MDC are measured.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 75, asecond CDR sequence consisting of SEQ ID NO: 76, and a third CDRsequence consisting of SEQ ID NO:65; and b) the light chain variabledomain comprises first CDR sequence consisting of amino acid sequenceSEQ ID NO: 77, a second CDR sequence consisting of SEQ ID NO: 78, and athird CDR sequence consisting of SEQ ID NO:68. In an embodiment, theantibody is used to treat diseases including atopic dermatitis, contactdermatitis, drug-induced allergic reactions, skin-tropic viruses andviral associated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis, and bullouspemphigoid, as discussed herein. In another embodiment, chemokines suchare TARC or MDC are measured.

In an embodiment, the present invention pertains to an isolated antibodyas disclosed herein wherein a) the heavy chain variable domain comprisesfirst CDR sequence consisting of amino acid sequence SEQ ID NO: 80, asecond CDR sequence consisting of SEQ ID NO: 817, and a third CDRsequence consisting of SEQ ID NO:82; and b) the light chain variabledomain comprises first CDR sequence consisting of amino acid sequenceSEQ ID NO: 83, a second CDR sequence consisting of SEQ ID NO: 84, and athird CDR sequence consisting of SEQ ID NO:85. In an embodiment, theantibody is used to treat diseases including atopic dermatitis, contactdermatitis, drug-induced allergic reactions, skin-tropic viruses andviral associated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis, and bullouspemphigoid, as discussed herein. In another embodiment, chemokines suchare TARC or MDC are measured.

Within one aspect, the invention provides a monoclonal antibody orantibody fragment that competes for specifically binding to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 whereinthe monoclonal antibody comprises a light chain variable region and aheavy chain variable region selected from the group consisting of: a) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 8 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 9; b) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 10 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 11; c) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:12 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 13; d) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 14 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 15; e) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 16 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 17; f) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 18 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 19; g) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 20 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:21; h) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 22 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 23; i) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 24 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 25; andi) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 and wherein the monoclonal antibody orantibody fragment is used in conjunction with an human IgG4 Fc molecule.Within an embodiment, the monoclonal antibody or antibody fragmentinhibits, blocks, or neutralizes the interaction of IL-31 (SEQ ID NO:2)with IL-31RA (SEQ ID NO:5). Within another embodiment, the monoclonalantibody or antibody fragment is selected from the group consisting of:(a) a murine monoclonal antibody or antibody fragment; (b) a humanizedantibody or antibody fragment or antibody fragment; and (c) a humanmonoclonal antibody. Within another embodiment, the antibody furthercomprises PEGylation.

Within another aspect, the invention provides, a monoclonal antibody orantibody fragment comprising a light chain variable region and a heavychain variable region where the monoclonal antibody or antibody fragmentis selected from the group consisting of: a) a monoclonal antibody orantibody fragment that competes for specifically binding to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 whereinthe monoclonal antibody or antibody fragment comprises a light chainvariable region and a heavy chain variable region selected from thegroup consisting of: i) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 8 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 9; and ii) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:26 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 27; and b) a monoclonal antibody or antibody fragment thatcompetes for specifically binding to a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 wherein the monoclonal antibody orantibody fragment comprises a light chain variable region and a heavychain variable region selected from the group consisting of: i) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:10 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 11; ii) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 12 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13; iii) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 14 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 15; iv) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 16 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 17; v) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 18 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:19; vi) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 20 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 21; vii) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 22 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 23; andvii) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 24 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 25 and wherein the monoclonal antibody orantibody fragment is used in conjunction with an human IgG4 Fc molecule.Within an embodiment, the monoclonal antibody or antibody fragmentinhibits, blocks, or neutralizes the interaction of IL-31 (SEQ ID NO:2)with IL-31RA (SEQ ID NO:5). Within another embodiment the monoclonalantibody or antibody fragment competes for specifically binding to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 whereinthe monoclonal antibody or antibody fragment comprises a light chainvariable region and a heavy chain variable region selected from thegroup consisting of: a) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 8 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 9; and b) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 26 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 27 and wherein the monoclonal antibody or antibody fragment isused in conjunction with an human IgG4 Fc molecule. Within anotherembodiment, the monoclonal antibody is selected from the groupconsisting of: (a) a murine monoclonal antibody or antibody fragment;(b) a humanized antibody or antibody fragment or antibody fragment; and(c) a human monoclonal antibody. Within another embodiment, the antibodyfurther comprises PEGylation. Within another embodiment, the monoclonalantibody or antibody fragment competes for specifically binding to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 whereinthe monoclonal antibody or antibody fragment comprises a light chainvariable region and a heavy chain variable region selected from thegroup consisting of: a) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 10 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 11; b) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 12 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 13; c) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 14 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 15; d) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 16 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:17; e) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 18 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 19; f) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 20 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 21; g)a light chain variable region comprising the amino acid sequence of SEQID NO: 22 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 23; and h) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 24 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 25.Within another embodiment, the monoclonal antibody is selected from thegroup consisting of: (a) a murine monoclonal antibody or antibodyfragment; (b) a humanized antibody or antibody fragment or antibodyfragment; and (c) a human monoclonal antibody. Within anotherembodiment, the antibody further comprises PEGylation.

Within another aspect, the invention provides, a method of reducing,blocking, inhibiting, or neutralizing inflammation in a mammalcomprising administering to the mammal an monoclonal antibody orantibody fragment that competes for specifically binding to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 whereinthe monoclonal antibody comprises a light chain variable region and aheavy chain variable region selected from the group consisting of: a) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 8 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 9; b) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 10 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 11; c) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:12 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 13; d) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 14 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 15; e) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 16 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 17; f) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 18 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 19; g) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 20 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:21; h) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 22 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 23; i) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 24 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 25; andi) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 and wherein the monoclonal antibody orantibody fragment is used in conjunction with an human IgG4 Fc molecule.Within an embodiment, administration of the antibody to the mammalreduces, blocks, inhibits, or neutralizes production of pro-inflammatorychemokines. Within a further embodiment, the pro-inflammatory chemokinesare TARC or MDC. Within another embodiment, the monoclonal antibody orantibody fragment competes for specifically binding to a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 wherein themonoclonal antibody or antibody fragment comprises a light chainvariable region and a heavy chain variable region selected from thegroup consisting of: a) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 8 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 9; and b) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 26 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 27 and wherein the monoclonal antibody or antibody fragment isused in conjunction with an human IgG4 Fc molecule. Within anotherembodiment, the monoclonal antibody or antibody fragment competes forspecifically binding to a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2 wherein the monoclonal antibody or antibody fragmentcomprises a light chain variable region and a heavy chain variableregion selected from the group consisting of: a) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 10 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:11; b) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 12 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 13; c) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 15; d)a light chain variable region comprising the amino acid sequence of SEQID NO: 16 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17; e) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 18 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 19; f) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:20 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 21; g) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 22 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 23; and h) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:24 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 25.

Within another aspect, the invention provides, a method of reducing,blocking, inhibiting, or neutralizing pruritis in a mammal comprisingadministering to the mammal an monoclonal antibody or antibody fragmentthat competes for specifically binding to a polypeptide comprising theamino acid sequence of SEQ ID NO: 2 wherein the monoclonal antibodycomprises a light chain variable region and a heavy chain variableregion selected from the group consisting of: a) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 8 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:9; b) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 10 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 11; c) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 12 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 13; d)a light chain variable region comprising the amino acid sequence of SEQID NO: 14 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 15; e) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 16 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 17; f) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:18 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 19; g) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 20 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 21; h) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 22 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 23; i) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 24 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 25; and i) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 26 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:27 and wherein the monoclonal antibody or antibody fragment is used inconjunction with an human IgG4 Fc molecule. Within an embodiment, themonoclonal antibody or antibody fragment competes for specificallybinding to a polypeptide comprising the amino acid sequence of SEQ IDNO: 2 wherein the monoclonal antibody or antibody fragment comprises alight chain variable region and a heavy chain variable region selectedfrom the group consisting of: a) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 8 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 9; andb) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 and wherein the monoclonal antibody orantibody fragment is used in conjunction with an human IgG4 Fc molecule.Within another embodiment the monoclonal antibody or antibody fragmentcompetes for specifically binding to a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 wherein the monoclonal antibody orantibody fragment comprises a light chain variable region and a heavychain variable region selected from the group consisting of: a) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:10 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 11; b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 12 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13; c) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 14 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 15; d) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 16 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 17; e) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 18 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:19; f) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 20 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 21; g) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 22 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 23; andh) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 24 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 25. Within another embodiment,administration of the monoclonal antibody or antibody fragment reduces,blocks, inhibits, or neutralizes dermatitis. Within a furtherembodiment, the dermatitis is atopic dermatitis or prurigo nodularis.

Within another aspect, the invention provides a method of reducing,blocking, inhibiting, or neutralizing scratching in a mammal comprisingadministering to the mammal an monoclonal antibody or antibody fragmentthat competes for specifically binding to a polypeptide comprising theamino acid sequence of SEQ ID NO: 2 wherein the monoclonal antibodycomprises a light chain variable region and a heavy chain variableregion selected from the group consisting of: a) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 8 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:9; b) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 10 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 11; c) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 12 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 13; d)a light chain variable region comprising the amino acid sequence of SEQID NO: 14 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 15; e) a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 16 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 17; f) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:18 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 19; g) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 20 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 21; h) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 22 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 23; i) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 24 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 25; and j) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 26 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:27 and wherein the monoclonal antibody or antibody fragment is used inconjunction with an human IgG4 Fc molecule. Within another embodimentthe monoclonal antibody or antibody fragment competes for specificallybinding to a polypeptide comprising the amino acid sequence of SEQ IDNO: 2 wherein the monoclonal antibody or antibody fragment comprises alight chain variable region and a heavy chain variable region selectedfrom the group consisting of: a) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 8 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 9; andb) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 and wherein the monoclonal antibody orantibody fragment is used in conjunction with an human IgG4 Fc molecule.Within another embodiment, the monoclonal antibody or antibody fragmentcompetes for specifically binding to a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 wherein the monoclonal antibody orantibody fragment comprises a light chain variable region and a heavychain variable region selected from the group consisting of: a) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:10 and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 11; b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 12 and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13; c) a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 14 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 15; d) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 16 and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 17; e) a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 18 and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:19; f) a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 20 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 21; g) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 22 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 23; andh) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 24 and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 25.

Within another aspect, the present invention provides a method forinhibiting IL-31-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of an IL-31 binding molecules or IL-31 antagonistsas disclosed herein sufficient to reduce proliferation ordifferentiation of the hematopoietic cells in the bone marrow orperipheral blood cells as compared to bone marrow or peripheral bloodcells cultured in the absence of soluble cytokine receptor. In oneembodiment the method for inhibiting IL-31-induced proliferation ordifferentiation of hematopoietic cells and hematopoietic cellprogenitors is as disclosed above, wherein the hematopoietic cells andhematopoietic progenitor cells are lymphoid cells. In another embodimentthe method for inhibiting IL-31-induced proliferation or differentiationof hematopoietic cells and hematopoietic cell progenitors is asdisclosed above, wherein the lymphoid cells are macrophages or T cells.

Within another aspect, the present invention provides a method ofreducing IL-31-induced inflammation comprising administering to a mammalwith inflammation an amount of a composition of a an IL-31 bindingmolecules or IL-31 antagonists as disclosed herein sufficient to reduceinflammation.

Within another aspect, the present invention provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of an inflammatory molecule; (2)administering a composition comprising an IL-31 binding molecules orIL-31 antagonists as disclosed herein in an acceptable pharmaceuticalvehicle; (3) determining a post administration level of the inflammatorymolecule; (4) comparing the level of the inflammatory molecule in step(1) to the level of the inflammatory molecule in step (3), wherein alack of increase or a decrease the inflammatory molecule level isindicative of suppressing an inflammatory response. In one embodiment,the antibody is as disclosed above, wherein the antibody furthercomprises a radionuclide, enzyme, substrate, cofactor, fluorescentmarker, chemiluminescent marker, peptide tag, magnetic particle, drug,or toxin.

Within another aspect, the present invention provides a method forinhibiting IL-31-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of an IL-31 binding molecules or IL-31 antagonistsas disclosed herein sufficient to reduce proliferation ordifferentiation of the hematopoietic cells in the bone marrow orperipheral blood cells as compared to bone marrow or peripheral bloodcells cultured in the absence of soluble cytokine receptor. In oneembodiment the method for inhibiting IL-31-induced proliferation ordifferentiation of hematopoietic cells and hematopoietic cellprogenitors is as disclosed above, wherein the hematopoietic cells andhematopoietic progenitor cells are lymphoid cells. In another embodimentthe method for inhibiting IL-31-induced proliferation or differentiationof hematopoietic cells and hematopoietic cell progenitors is asdisclosed above, wherein the lymphoid cells are macrophages or T cells.

Within another aspect, the present invention provides a method ofreducing IL-31-induced inflammation comprising administering to a mammalwith inflammation an amount of a composition of a an IL-31 bindingmolecules or IL-31 antagonists as disclosed herein sufficient to reduceinflammation.

Within another aspect, the present invention provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of an inflammatory molecule; (2)administering a composition comprising an antibody IL-31 bindingmolecules or IL-31 antagonists as disclosed herein in an acceptablepharmaceutical vehicle; (3) determining a post administration level ofthe inflammatory molecule; (4) comparing the level of the inflammatorymolecule in step (1) to the level of the inflammatory molecule in step(3), wherein a lack of increase or a decrease in the inflammatorymolecule level is indicative of suppressing an inflammatory response.

Within another aspect, the present invention provides a method oftreating a mammal afflicted with an inflammatory disease in which IL-31plays a role, comprising: administering an antagonist of IL-31 to themammal such that the inflammation is reduced, wherein the antagonist isselected from the group consisting of an IL-31 binding molecules orIL-31 antagonists that specifically binds a polypeptide or polypeptidefragment of IL-31 (SEQ ID NO:2). In one embodiment, the method oftreating a mammal afflicted with an inflammatory disease is as disclosedabove, wherein the disease is a chronic inflammatory disease. In anotherembodiment, the method of treating a mammal afflicted with aninflammatory disease is as disclosed above, wherein the disease is achronic inflammatory disease selected from the group consisting of:inflammatory bowel disease; ulcerative colitis; Crohn's disease; atopicdermatitis; eczema; and psoriasis. In another embodiment, the method oftreating a mammal afflicted with an inflammatory disease is as disclosedabove, wherein the disease is an acute inflammatory disease. In anotherembodiment, the method of treating a mammal afflicted with aninflammatory disease is as disclosed above, wherein the disease is anacute inflammatory disease selected from the group consisting of:endotoxemia; septicemia; toxic shock syndrome; and infectious disease.In another embodiment, the method of treating a mammal afflicted with aninflammatory disease is as disclosed above, wherein the antibody furthercomprises a radionuclide, enzyme, substrate, cofactor, fluorescentmarker, chemiluminescent marker, peptide tag, magnetic particle, drug,or toxin.

Within another aspect, the present invention provides a method fordetecting inflammation in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an IL-31 binding molecules or IL-31 antagonists as disclosedherein under conditions wherein the IL-31 binding molecules or IL-31antagonists binds to its complementary polypeptide in the tissue orbiological sample; visualizing the IL-31 binding molecules or IL-31antagonists bound in the tissue or biological sample; and comparinglevels of IL-31 binding molecules or IL-31 antagonists bound in thetissue or biological sample from the patient to a normal control tissueor biological sample, wherein an increase in the level of IL-31 bindingmolecules or IL-31 antagonists bound to the patient tissue or biologicalsample relative to the normal control tissue or biological sample isindicative of inflammation in the patient.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Determination of Variable Regions

Sequences of the light and heavy chain variable regions were determinedin the following manner:

RNA Extraction/5′ RACE Ready cDNA Production:

Hybridoma cell lines (approximately 4.5×106 cells) were collected bycentrifugation after washing in 1×PBS. RNA was purified using Qiagen'sRNeasy mini purification kit according to the manufacture'sinstructions. Resulting RNA was displayed on a 1.2% E-Gel forvalidation. First strand cDNA synthesis was performed using BDBiosciences BD SMART RACE cDNA Amplification Kit, which provided 5′ RACEReady cDNA.

Amplification of Light and Heavy Chain Variable Region Sequences:

5′ RACE ready cDNA was used as template for PCR as described in BDBiosciences BD SMART RACE cDNA Amplification Kit. Both the heavy andlight chain PCR amplifications used the 10×UPM (Universal Primer Mix)provided by the kit as the 5′ PCR oligonucleotide. The 3′ PCRoligonucleotides were as follows;

Mouse kappa (zc54289: 5′-CGACTGAGCCACCTCCAGATG TTAACTGCTCAC-3′SEQ ID NO: 28) Mouse IgG1 (zc54983: 5′-CAGGGGCCAGTGGATAGACAGA TGGGGG-3′SEQ ID NO: 29) Moue IgG2a (zc55640: 5′-CAGGGGCCAGTGGATAGACCGA TGGGG-3′SEQ ID NO: 30)

The light and heavy chain variable region sequence PCR products were gelpurified using GE Healthcare illustra GFX tm PCR DNA and Gel BandPurification Kit and cloned via Invitrogen TOPO TA Cloning Kit. Eightindividual colonies per hybridoma per variable region were screened bycolony PCR with M13R and M13F kit primers and submitted for sequencing.Sequencing was performed using ABI PRISM BigDye Terminator v3.0 CycleSequencing Kit (Applied Biosystems, Foster City, Calif.). Sequencingreactions were purified using EdgeBioSystems Centriflex Gel FiltrationCartridges (Gaithersburg, Md.) and run on an ABI PRISM 377 DNA Sequencer(Applied Biosystems, Foster City, Calif.). Resultant sequence data wasassembled and edited using Sequencher v4.1 software (GeneCodesCorporation, Ann Arbor, Mich.)

Table 1 shows the SEQ ID NO:s for the sequences of the variable regions,which are further described in FIGS. 1-4.

TABLE 1 Sequence Listing Numbers for Variable Light and Variable HeavyRegions Heavy Chain Light Chain Variable Variable Region Light ChainRegion with signal Heavy Chain with signal Clone Variable Regionsequence Variable Region sequence Number SEQ ID NO: SEQ ID NO: SEQ IDNO: SEQ ID NO: 292.12.3.1  8 31  9 32 292.84.1.6 8 with Arg 31 with Arg9 with 32 with substituted at substituted at position substitutions:substitutions: position 42 62 Thr at position Thr at position 50; 50;Ser at position Ser at position 69; 88; Asn at position Asn at position77; and 95; and Phe at position Phe at position 95 114 292.63.5.3 10 3311 34 294.144.3.5 12 35 13 36 292.39.5.3 14 37 15 38 292.51.5.2 16 39 1740 292.64.6.5.5 18 41 19 42 292.105.4.1 20 43 21 44 292.109.4.4 22 45 2346 292.118.6.4 24 47 25 48 292.72.3.1 26 49 27 50

Table 2 shows the SEQ ID NO:s for the sequences of the variable regions,which are further described in FIGS. 1-4.

TABLE 2Sequence Listing Numbers for CDRs of Variable Light and Variable Heavy RegionsVariable Variable Variable Variable Variable Variable Clone Heavy HeavyHeavy Light Light Light Number CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 292.12.3.1SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 51 NO: 52 NO: 53 NO: 54NO: 55 NO: 56 (CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of(CDR2 VL of (CDR3 VL of 292.12.3.1): 292.12.3.1): 292.12.3.1):292.12.3.1): 292.12.3.1): 292.12.3.1): RYWMQ AIYPGDGDT PDGYYAAPYRASGNIHNYLA NAKTLAD QHFWSTPWT RYSQKFKG GMDY 292.84.1.6 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: 51 NO: 57 NO: 53 NO: 54 NO: 55 NO: 56(CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of (CDR2 VL of (CDR3 VL of292.12.3.1): 292.84.1.6): 292.12.3.1): 292.12.3.1): 292.12.3.1):292.12.3.1): RYWMQ TIYPGDGDT PDGYYAAPY RASGNIHNYLA NAKTLAD QHFWSTPWTRYSQKFG GMDY 292.72.3.1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 51NO: 58 NO: 59 NO: 60 NO: 61 NO: 62 (CDR1 VH of (CDR2 VH of (CDR3 VH of(CDR1 VL of (CDR2 VL of (CDR3 VL of 292.12.3.1): 292.72.3.1):292.72.3.1): 292.72.3.1): 292.72.3.1): 292.72.3.1): RYWMQ AIYPRDGDTPDGSYAAPN RASGSIHNYLA NAETLAD QHFWITPWT RYSQKFKG GMEY 292.63.5.3 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 63 NO: 64 NO: 65 NO: 66 NO: 67NO: 68 (CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of (CDR2 VL of(CDR3 VL of 292.63.5.3): 292.63.5.3): 292.63.5.3): 292.63.5.3):292.63.5.3): 292.63.5.3): TFIMS TINSGGYYT QEGWSSAYF KSSQSLLNGS FASTRDSQQHYDTPYT FHPDSVKG SY NQKNYLA 292.39.5.3 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: 69 NO: 70 NO: 71 NO: 72 NO: 73 NO: 74 (CDR1 VH of(CDR2 VH of (CDR3 VH of (CDR1 VL of (CDR2 VL of (CDR3 VL of 292.39.5.3):292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3): TYIMSTINSGGYYT QEGWSSAW NSSQSLLNSSN FASTGES QQHFSTPYT LYPDSVKG FAY QKNYLA292.51.5.2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 75 NO: 76NO: 65 NO: 77 NO: 78 NO: 68 (CDR1 VH of (CDR2 VH of (CDR3 VH of(CDR1 VL of (CDR2 VL of (CDR3 VL of 292.51.5.2): 292.51.5.2):292.63.5.3): 292.51.5.2): 292.51.5.2): 292.63.5.3): SFVMS TINSGGYYSQEGWSSAYF KSSQSLLNSSN FTSTRES QQHYDTPYT FHPDSVKG SY QKNYLA 292.64.6.SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 5.5 NO: 69 NO: 70 NO: 71NO: 72 NO: 73 NO: 74 (CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of(CDR2 VL of (CDR3 VL of 292.39.5.3): 292.39.5.3): 292.39.5.3):292.39.5.3): 292.39.5.3): 292.39.5.3): TYIMS TINSGGYYT QEGWSSAWNSSQSLLNSSN FASTGES QQHFSTPYT LYPDSVKG FAY QKNYLA 292.105.4.1 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 69 NO: 70 NO: 71 NO: 72 NO: 73NO: 74 (CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of (CDR2 VL of(CDR3 VL of 292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3):292.39.5.3): 292.39.5.3): TYIMS TINSGGYYT QEGWSSAW NSSQSLLNSSN FASTGESQQHFSTPYT LYPDSVKG FAY QKNYLA 292.109.4.4 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: 69 NO: 70 NO: 71 NO: 72 NO: 73 NO: 74 (CDR1 VH of(CDR2 VH of (CDR3 VH of (CDR1 VL of (CDR2 VL of (CDR3 VL of 292.39.5.3):292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3): TYIMSTINSGGYYT QEGWSSAW NSSQSLLNSSN FASTGES QQHFSTYPT LYPDSVKG FAY QKNYLA292.118.6.4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 69 NO: 79NO: 71 NO: 72 NO: 73 NO: 74 (CDR1 VH of (CDR2 VH of (CDR3 VH of(CDR1 VL of (CDR2 VL of (CDR3 VL of 292.39.5.3): 292.118.6.4):292.39.5.3): 292.39.5.3): 292.39.5.3): 292.39.5.3): TYIMS TINSGGYYTQEGWSSAW NSSQSLLNSSN FASTGES QQHFSTPYT IYPDSVKG FAY QKNYLA 294.144.3.5SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 80 NO: 81 NO: 82 NO: 83NO: 84 NO: 85 (CDR1 VH of (CDR2 VH of (CDR3 VH of (CDR1 VL of(CDR2 VL of (CDR3 VL of 292.144.3.5): 292.144.3.5): 292.144.3.5):292.144.3.5): 292.144.3.5): 292.144.3.5): TYWIE EILPGRGTT ESKLGDDDYSASSSVSYMH DTTKLAS FQGSEHPLT NYNAKFQG

Hybridomas, 292.12.3.1 and 292.84.1.6, express light and heavy chainsthat share extensive sequence identity with each other. The amino acidsequence alignments of the 292.12.3.1 and 292.84.1.6 light chain andheavy chain variable regions are shown in FIG. 2. The high degree ofshared sequence identity suggests that the light chain variable regionsare derived from the same light chain variable region germline gene,while the heavy chain variable regions are also derived from the sameheavy chain variable region germline gene. Additionally, the identitythroughout both the light and heavy chain CDR3 and FR4 indicateutilization of the same JL in the light chain, and the same JH and Dregions as well as the N and P nucleotide additions in the heavy chainCDR3. Both hybridomas 292.12.3.1 and 292.84.1.6 express kappa lightchains, but 292.12.3.1 expresses an IgG1 heavy chain while 292.84.1.6expresses an IgG2a heavy chain. It appears that both of these hybridomasare derivatives of the same initial B cell immunoglobulin locirearrangement events and that 292.84.1.6 is a result of a subsequentclass switch to an IgG2a heavy chain. Either prior to, or after, theclass switch, 292.12.3.1 and 292.84.1.6 diverged by the incorporation offurther somatic mutations which led to the single amino acid differencein the light chain and the 4 amino acid differences in the heavy chain.

Example 2 Amino Terminal Protein Sequence Determination

N-terminal protein sequencing was used to determine heavy and lightchain antibody sequences. The antibodies were processed with and withoutpyroglutumate amino peptidase (PGAP) treatment. Untreated samples wereprocessed by addition of 100 picomoles (pmol) protein and water. PGAPtreated samples were processed by addition of 100 pmol protein, water,0.03% SDS, 5×PGAP buffer (Takara Bio Inc., Japan) and PGAP enzyme (1 mU)in 1×PGAP buffer. The PGAP reaction was run for 10 minutes at 95° C.This enzymatic treatment removed the N-terminally blocking pyroglutamicacid groups. Reducing SDS PAGE buffer was added to both the untreatedand PGAP treated samples and then the samples were heated for 5 minutesin a boiling water bath. The samples were run on a SDS PAGE gradientgel. The gel was transferred to a PVDF membrane and stained withcoomassie blue. Two visible bands were observed for each sample withapparent SDS PAGE molecular weights of 50 kDa and 25 kDa. Each band wasexcised and subjected to N-terminal protein sequencing. Twenty sequencecycles were run to determine the sequence.

Example 3 Luciferase Assay on Human Transformed Epithelial Cell LinesVia Transient Infection with an Adenoviral STAT/SRE Reporter Gene

Inhibition, reduction, and/or neutralization of IL-31 activity can bemeasured by the luciferase assay. For example, human transformed celllines can be seeded in 96-well flat-bottom plates at 10,000 cell/well inregular growth media as specified for each cell type. The following day,the cells are infected with an adenovirus reporter construct, KZ136, ata multiplicity of infection of 5000. The KZ136 reporter contains theSTAT elements in addition to a serum response element. The total volumeis 100 ul/well using DMEM supplemented with 2 mM L-glutamine (GibcoBRL),1 mM Sodium Pyruvate (GibcoBRL) and 1× Insulin-Transferrin-Seleniumsupplement (GibcoBRL) (hereinafter referred to as serum-free media).Cells are cultured overnight.

The following day, the media is removed and replaced with 100 μl ofinduction media. The induction media is human IL-31 diluted inserum-free media at 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25ng/ml, 3.125 ng/ml and 1.56 ng/ml. A positive control of 20% FBS is usedto validate the assay and to ensure the infection by adenovirus issuccessful. The cells are induced for 5 hours at which time the media isaspirated. The cells are then washed in 50 μl/well of PBS, andsubsequently lysed in 30 μl/well of 1× cell lysis buffer (Promega).After a 10-minute incubation at room temperature, 25 μl/well of lysateis transferred to opaque white 96-well plates. The plates are then readon the Luminometer using 5-second integration with 40 μl/well injectionof luciferase substrate (Promega).

Example 4 IL-31 Bioassay

BAF3 cells transfected with hzCYTOR17 (IL-31RA), hOSMRB, and KZ134 aregrown to 5×10⁵ and 1×10⁶ cells/mL. Cells are washed with assay media(RPMI 1640, 10% FBS, L-Glutamine, Sodium Pyruvate, and Pen/Strep (allGibco)) and resuspended at 3×10⁵ cell/mL in assay medium. In a 96-wellopaque plate, hIL-31 standards are titered in duplicate from 600 pg/mLto 9.38 pg/mL in assay medium via a 100 μL/well, 1:2 serial dilution.Quality control standards are added in duplicate to the plate at 350pg/mL and 35 pg/mL in 100 μL. Test samples are often diluted 1:2 or 1:4and added in duplicate to the sample wells. 100 μL of the washed BAF3cells are added to each well for a final concentration of 3×10⁴cells/well. The plate is incubated for 16-24 hours at +37° C. in a 5%CO₂ incubator. The plate is centrifuged at 1200 RPM for 5 minutes, mediaflicked off and 25 μL/well of lysis buffer (Promega) added to each well.After 10 minutes the plate is read on a luminometer (Berthold). Theluminometer adds 40 μL/well of luciferase substrate mix (Promega) andintegrated the luminescence for a period of 4 seconds Luminescencevalues are exported to a spreadsheet where they are analyzed andconverted into picograms of IL-31 per 10⁶ cells per mL of volume.

Example 5 IL-31 Involvement in Initiation and Perpetuation of ContactHyper-Sensitivity In Vivo

Method I

BALB/c mice are painted on shaved mid-back with 25 ul of 0.5% DNFBdissolved (2,4, dinitro-fluoro-benzene, Sigma, St. Louis Mo.) inacetone:olive oil (4:1) solution using a pipettor. A vehicle controlgroup receives 25 ul of acetone:olive oil only. After 5 days, mice areanaesthetized with isofluorane in an inhalation chamber and both earpinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare then challenged by applying 10 ul of 0.25% DNFB in acetone:olive oil(4:1) to both sides of each ear of all mice. Contact hyper-sensitivityis measured at 24 h and 48 h later as the difference between the rightear (challenged) and the left ear (unchallenged). All measurements aredone with an engineer's micrometer. Background values are determined bythe difference in ear swelling between the challenged and unchallengedears of naive mice.

Whole blood and serum for FACS and/or ELISA analysis are collected priorto sacrifice and ears are collected for histology.

Method II (Induces Th2 Responses)

BALB/c mice are painted on shaved mid-back with 100 ul of 0.5% FITC(fluorescein isothiocyanate) in a 1:1 solution of acetone/dibutylphthalate (MSDS available using pipettor on days 1, 2 and 8. On day 13,mice are anaesthetized with isofluorane in an inhalation chamber andboth ear pinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare challenged by applying 25 ul of 0.5% FITC (in 1:1 acetone/dibutylphthalate) to the dorsal surface of each ear. Contact hyper-sensitivityis measured at 24 h and 48 h later as the difference between the rightear (challenged) and the left ear (unchallenged). All measurements aredone with an engineer's micrometer. Background values are determined bythe difference in ear swelling between the challenged and unchallengedears of naive mice. Whole blood and serum for FACS and/or ELISA analysisare collected prior to sacrifice and ears are collected for histology.

Method III (Induces Th1 Responses)

BALB/c mice are painted on shaved mid-back with 25 ul of 2% oxazalone(in 4:1 acetone/olive oil) using pipettor. On day 7, mice areanaesthetized with isofluorane in an inhalation chamber and both earpinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare challenged by applying 8 ul of oxazalone to the dorsal surface ofeach ear. Contact hyper-sensitivity is measured at 24 h and 48 h lateras the difference between the right ear (challenged) and the left ear(unchallenged). All measurements are done with an engineer's micrometer.Background values are determined by the difference in ear swellingbetween the challenged and unchallenged ears of naive mice. Whole bloodand serum for FACS and/or ELISA analysis are collected prior tosacrifice and ears are collected for histology.

Involvement of IL-31 in the initiation and perpetuation of contacthyper-sensitivity is tested using the IL-31 binding molecules or IL-31antagonists described herein against IL-31 both at the sensitization andchallenge phases of the experiment.

Example 6 IL-31 Involvement in Atopic Dermatitis In Vivo

Methods I (Sensitization of NC/Nga Mice)

4 weeks old male NC/Nga mice (CRL, Japan) are housed in SPF quarantineconditions for 4 weeks to acclimate. The mice are approximately 10-11weeks old at the start of the antigen sensitization. Mice areanaesthetized with isofluorane and backs rae shaved with electricclippers. Approximately 10 ug of Dermatophagoides pteronyssinus (Dp)(Indoor Biotechnologies, special order) extract is injectedintradermally at the nape of the neck 3 times per week for 5 to 6 weeksuntil mice developed skin lesions. Control animals receive 10 ul PBSintradermal injections 3 times per week. The Dp extract is preparedaccording to method by Matsuoka and colleagues. Matsuoka H., et al.,Allergy: 58, 139 (2003). Briefly, 595 mg Dp lyophilized spent cultureextract is dissolved in 12 mL sterile PBS (Gibco). Dp is mixed in a 50mL Falcon tube on a shaking rocker for 30 minutes. The extract is spunfor 10 minutes at 2000 rpm and the supernatant is collected andaliquoted into 1 mL cryovial tubes and stored at −20° C.

The effects of IL-31 binding molecules or IL-31 antagonists are measuredby inhibition of scratching, itching, and or dermatitis.

Methods II (Sensitization of DO11.10 Mice)

DO11.10 transgenic mice are bred from an in-house colony and are between9.5 and 14 weeks old at start of antigen sensitization. 24 hours priorto epicutaneous sensitization mice are anaesthetized with isofluoraneand the entire trunk (back and abdomen) of mice are shaved with electricclippers. The mice are then tape stripped with Elastin surgical tape(Johnson and Johnson) on the back. 1 cm2 sterile gauze patches arewetted with either 500 ug ovalbumin (Calbiochem 32467) or sterile PBS(Gibco) and adhered to left backside of mice with DuoDerm Extra ThinDressing (ConvaTec 187932). The patch and dressing are then covered in abody wrap of the Elastin surgical tape so mice could not remove ordestroy the patches. Patches are worn for 7 days and removed. The miceare rested for two weeks before having another round of epicutaneoussensitization. Mice receive a total of three one-week sensitizations.

The effects of IL-31 binding molecules or IL-31 antagonists are measuredby inhibition of scratching, itching, and or dermatitis and/or areduction in IL-31RA expression in kerotinocytes.

Example 7 Reduction of TARC and MDC in Response to Anti-Il-31 Antibodyin AD Mouse Models

Method I

Six-week old male NC/Nga mice (CRL Japan) are sensitized intradermallywith 50 μg dust mite extract (D. pteronyssinus, Indoor Biotechnologies)three times a week on the back and scored for AD-like lesions. After 5weeks of sensitization the mice are euthanized and the right ears wereexcised and placed into a single well of a 48-well culture dish(Corning) supplemented with RPMI+2% FBS (GIBCO Invitrogen). Plates areplaced in 5% CO2 humidity controlled incubators. Supernatants arecollected after 24 hours and frozen at −20° C. until further analysis.

Method II

Twelve-week old female NC/Nga mice (CRL Japan) are sensitizedintradermally with 10 μg SEB (Toxin Technology) in the ear and on theback three times per week. The mice are scored for AD-like lesions.After 5 weeks of sensitization the mice are euthanized and 6 mm biopsypunches were taken from the injected ear of each mouse and placed into asingle well of a 48-well culture dish supplemented with RPMI+2% FBS.Plates were placed in 5% CO2 humidity controlled incubators.Supernatants are collected after 24 hours and frozen at −20° C. untilfurther analysis.

Groups of mice in both studies are treated with IL-31 binding moleculesor IL-31 antagonists intraperitoneally two times each week startingafter 1 to 2 weeks of sensitization.

TARC and MDC concentrations in the 24-hour supernatant samples aremeasured by conventional ELISA (R&D Systems).

Example 8 Administration of IL-31 Neutralizing Antibody

Normal female BALB/c mice (CRL) approximately 8 to 12 weeks old areimplanted subcutaneously with 14-day osmotic pumps (Alzet, #2002)delivering 1 ug/day mIL-31. Groups of mice receive intraperitoneal(i.p.) injections of rat anti-mouse IL-31 monoclonal antibody 10 mg/kg(200 ug/mouse) twice weekly starting 1 week prior to IL-31 delivery.Control groups of mice receive i.p. injections of vehicle (PBS/0.1% BSA)with the identical dosing schedules. Mice are scored daily for alopeciaand pruritis using the following criteria: 0=no scratching, animalappears normal, 1=thinning of coat in small areas, scratching noted,2=minor hair loss (small patches), scratching, 3=moderate hair loss,scratching, and 4=severe hair loss, excessive scratching.

The effects of IL-31 binding molecules or IL-31 antagonists are measuredby a delay in onset of symptoms of approximately 5 to 7 days and a loweroverall score for alopecia and pruritis.

Example 9 Expression of Recombinant Chimeric Anti-Human IL-31 MonoclonalAntibodies

The heavy and light chain variable region sequences from two separatemouse anti-human IL31 monoclonal antibodies, 292.12.3.1 and 292.63.5.3,were obtained by PCR. The DNA sequences were determined and expressionconstructs were generated utilizing human constant region DNA sequences.

The light chain expression constructs consisted of a hybrid MPSV/CMVpromoter/enhancer directing expression of the chimeric mouse anti-humanIL31 variable region fused to a human immunoglobulin kappa constantregion.

The heavy chain expression constructs consisted of a hybrid MPSV/CMVpromoter/enhancer directing expression of the chimeric mouse anti-humanIL31 variable region fused to a human immunoglobulin IgG4 constantregion with an amino acid substitution in the hinge region, Serine 228changed to Proline.

The light and heavy chain expression constructs encoding the chimericlight and heavy chains from each hybridoma were co-transfected into HEK293F cells. Conditioned media was harvested after 4 days. Western blotanalysis demonstrated intact chimeric antibody of expected size onnon-reducing SDS-PAGE.

The antigen binding ability of the chimeric antibodies were determinedby an ELISA based protocol to measure apparent EC50 (effectiveconcentration to bind 50% of antigen at a fixed concentration). Theassay format utilized a goat anti-human Fc immobilization for capture ofhuman monoclonal antibodies from unprocessed cell culture conditionedmedia. A dilution series of biotinylated IL31 tested the “C” parameterof a 4-parameter fit which results in the apparent Kd (or EC50) ineither ng/mL or nM of IL31. The assay sensitivity is high enough thatdilute monoclonal antibody or chimeric antibody cell culture conditionedmedia can be evaluated. The Kd determined by this method generallyapproaches the Kd measured for purified, homogeneous monoclonalantibodies measured using Biacore. Both chimeric anti-IL31 antibodiesshowed similar EC50 values compared to the control “parental” mousehybridoma monoclonal antibody 292.63.5.3 as shown in Table 3.

TABLE 3 EC50 Determination of Conditioned Media from HEK 293F TransientTransfections Sample EC50 (ng/mL)^(a) EC50 (nM)^(a) Chimeric 292.63.5.31.3 0.08 Chimeric 292.12.3.1 2.0 0.12 Non-transfected HEK 293F Nobinding No binding control medium 292.63.5.3 (Lot E9289) 4.3 0.26Control monoclonal antibody ^(a)Average from duplicate measure

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An isolated monoclonal antibody that specificallybinds to a polypeptide consisting of amino acid residues 27-164 of SEQID NO:2, wherein the monoclonal antibody is a humanized antibody derivedfrom the hybridoma deposited with the American Type Culture Collectionhaving the ATCC Patent Deposit Designation PTA-6815, and wherein themonoclonal antibody comprises a human IgG4 heavy chain immunoglobulinconstant domain having a serine to proline mutation at Kabat position241.
 2. An isolated polynucleotide encoding a monoclonal antibody thatbinds to a polypeptide consisting of amino acid residues 27-164 of SEQID NO:2, wherein the monoclonal antibody is a humanized antibody derivedfrom the hybridoma deposited with the American Type Culture Collectionhaving the ATCC Patent Deposit Designation PTA-6815, and wherein themonoclonal antibody comprises a human IgG4 heavy chain immunoglobulinconstant domain having a Serine to Proline mutation at Kabat position241.
 3. An expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment comprising thepolynucleotide of claim 2; and a transcription terminator.
 4. A culturedcell comprising the expression vector of claim 3, wherein the cellexpresses the monoclonal antibody encoded by the DNA segment.
 5. Thecultured cell according to claim 4, wherein the cell is a eukaryoticcell.
 6. The cultured cell according to claim 5, wherein the eukaryoticcell is a Chinese Hamster Ovary (CHO) cell.
 7. A method of producing amonoclonal antibody comprising: culturing a cell comprising theexpression vector of claim 2; and recovering the expressed monoclonalantibody.
 8. A method of reducing IL-31-induced pruritis in a mammalcomprising administering to the mammal a therapeutically effectiveamount of the monoclonal antibody according to claim 1, wherein afteradministration the pruritis is reduced.
 9. A method of reducingIL-31-induced inflammation in a mammal comprising administering to themammal a therapeutically effective amount of the monoclonal antibodyaccording to claim 1, wherein after administration the inflammation isreduced.
 10. A method of reducing IL-31-induced scratching in a mammalcomprising administering to the mammal a therapeutically effectiveamount of the monoclonal antibody according to claim 1, wherein afteradministration the scratching is reduced.
 11. A method of treating apruritic disease in a mammal comprising administering to the mammal atherapeutically effective amount of the monoclonal antibody according toclaim 1, wherein the pruritic disease is selected from the groupconsisting of atopic dermatitis, contact dermatitis, drug-inducedallergic reactions, skin-tropic viruses and viral associated pruritis,vitiligo, cutaneous T cell lymphoma, alopecia aerata, acne rosacea, acnevulgaris, prurigo nodularis and bullous pemphigoid.
 12. A method oftreating a pruritic disease in a mammal comprising administering to themammal a therapeutically effective amount of a pharmaceuticalcomposition comprising the monoclonal antibody according to claim 1 anda pharmaceutically acceptable carrier, wherein the pruritic disease isselected from the group consisting of atopic dermatitis, contactdermatitis, drug-induced allergic reactions, skin-tropic viruses andviral associated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis and bullouspemphigoid.